Proteins associated with cell proliferation

ABSTRACT

The invention provides three human proteins associated with cell proliferation, referred to collectively as &#34;APOP&#34; and individually as &#34;APOP-1&#34;, &#34;APOP-2&#34;, and &#34;APOP-3&#34;, and polynucleotides which identify and encode APOP. The invention also provides expression vectors, host cells, agonists, antibodies and antagonists. The invention also provides methods for preventing and treating disorders associated with expression of APOP.

FIELD OF THE INVENTION

This invention relates to nucleic acid and amino acid sequences of threehuman proteins associated with cell proliferation and to the use ofthese sequences in the diagnosis, prevention, and treatment of disordersassociated with abnormal cell proliferation and apoptosis.

BACKGROUND OF THE INVENTION

Differentiation, growth, and function of eukaryotic cells arecoordinated with various mechanisms which regulate DNA replication andprevent excessive proliferation. While uncoordinated cell proliferationcan cause cancer, autoimmune diseases, and inflammatory diseases,programmed cell death or apoptosis removes excessive or damaged cellswithout causing tissue destruction and inflammatory response. A plethoraof genes, named oncogenes, have been identified with cancer. Severalhighly conserved processes have been shown to regulate apoptosis.

bcl-2 proto-oncogene is a repressor of apoptosis and functionsdownstream in the regulation of cell-death processes. The C. eleganshomolog of Bcl-2, CED-9, represses the apoptosis of 131 cells during thenematode development (Hengartner, M. O. and Horvitz, H. R. (1994) Cell76: 665-676). Several Bcl-2-related proteins, such as Bax, Bcl-X_(L),and Bad, share homology with Bcl-2 mostly within two conserved regions,named BH1 and BH2. Bcl-X_(L) and _(Bcl-2) are shown to repressapoptosis, while Bax and Bad promote apoptosis. Specifically, Bax iscapable of dimerizing with Bcl-2 and Bcl-X_(L), whereas Bad is able todimerize with Bcl-X_(L) but not with Bcl-2. When Bax dimerizes withBcl-2, the apoptosis-inhibiting function of Bcl-2 is suppressed (Oltvai,Z. N. et al. (1993) Cell 74: 609-619; Yin, X.-M. et al. (1994) Nature369: 321-323). Similarly, when Bad displaces Bax and dimerizes withBcl-X_(L), the apoptosis-repressive activity of Bcl-X_(L) is inhibited(Yang, E. et al. (1995) Cell 80: 285-291).

Leucine-rich repeat (LRR) is a structural motif of about 20 to 29 aminoacid residues in length associated with protein-protein interactions.The motif contains leucine or other aliphatic residues at positions 2,5, 7, 12, 16, 21, and 24 and asparagine, cysteine or threonine atposition 10. X-ray structure determination of LRR motifs suggests thateach LRR is composed of a β-sheet and an α-helix.

p37NB is a 37 kDa LRR protein identified in human neuroblastoma cells(Kim, D. et al. (1996) Biochim. Biophys. Acta 1309: 183-188). Northernblot hybridization and RT-PCR studies show that p37NB is differentiallyexpressed in several neuroblastoma cell lines. A related LRR protein,PRELP, is characterized as a 42 kDa secreted protein (Bengtsson, E. etal. (1995) J. Biol. Chem. 270: 25639-25644). PRELP consists of 10 LRRmotifs ranging in length from 20 to 26 residues with asparigine atposition 10. Northern analysis shows differential expression of PRELP invarious tissues.

MA-3 or TIS is a mouse protein associated with apoptosis (Shibahara, K.et al. (1995) Gene 166: 297-301; Onishi, Y. and Kizaki, H. (1996)Biochim. Biophys. Res. Commun. 228: 7-13). The nucleotide sequence ofthe mouse proteins predicts an amino acid sequence of 469 residues. MA-3is highly expressed in thymus and is present in all apoptosis-induciblecell lines including thymocytes, T cells, B cells, and pheochromocytoma(Shibahara et al, supra). TIS expression is down-regulated in the RVClymphoma cells incubated with an topoisomerase I inhibitor, an antitumordrug, and the low expression level of TIS may be a contributing factorto the cytotoxicity of the topoisomerase inhibitors (Onishi and Kizaki,supra).

The discovery of three new human proteins associated with cellproliferation and the polynucleotides encoding them satisfies a need inthe art by providing new compositions which are useful in the diagnosis,prevention, and treatment of disorders associated with abnormal cellproliferation and apoptosis.

SUMMARY OF THE INVENTION

The invention features three substantially purified polypeptides,proteins associated with cell proliferation, referred to collectively as"APOP" and individually as "APOP-1", "APOP-2", and "APOP-3." In oneaspect, the invention provides a substantially purified polypeptide,APOP, comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, a fragment of SEQID NO:1, a fragment of SEQ ID NO:3, and a fragment of SEQ ID NO:5.

The invention further provides a substantially purified variant of APOPhaving at least 90% amino acid identity to the amino acid sequences ofSEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5, or to a fragment of either ofthese sequences. The invention also provides an isolated and purifiedpolynucleotide sequence encoding the polypeptide comprising an aminoacid sequence selected from the group consisting of SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:5, a fragment of SEQ ID NO:1, a fragment of SEQ ID NO:3,and a fragment of SEQ ID NO:5. The invention also includes an isolatedand purified polynucleotide variant having at least 90% polynucleotideidentity to the polynucleotide sequence encoding the polypeptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, a fragment of SEQ ID NO:1, afragment of SEQ ID NO:3, and a fragment of SEQ ID NO:5.

Additionally, the invention provides a composition comprising apolynucleotide sequence encoding the polypeptide comprising the aminoacid sequence selected from the group consisting of SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:5, a fragment of SEQ ID NO:1, a fragment of SEQ ID NO:3,and a fragment of SEQ ID NO:5. The invention further provides anisolated and purified polynucleotide sequence which hybridizes understringent conditions to the polynucleotide sequence encoding thepolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, a fragment of SEQID NO:1, a fragment of SEQ ID NO:3, and a fragment of SEQ ID NO:5, aswell as an isolated and purified polynucleotide sequence which iscomplementary to the polynucleotide sequence encoding the polypeptidecomprising the amino acid sequence selected from the group consisting ofSEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, a fragment of SEQ ID NO:1, afragment of SEQ ID NO:3, and a fragment of SEQ ID NO:5.

The invention also provides an isolated and purified polynucleotidesequence comprising a polynucleotide sequence selected from the groupconsisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, a fragment of SEQID NO:2, a fragment of SEQ ID NO:4, and a fragment of SEQ ID NO:6. Theinvention further provides an isolated and purified polynucleotidevariant having at least 90% polynucleotide identity to thepolynucleotide sequence comprising a polynucleotide sequence selectedfrom the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, afragment of SEQ ID NO:2, a fragment of SEQ ID NO:4, and a fragment ofSEQ ID NO:6, as well as an isolated and purified polynucleotide sequencewhich is complementary to the polynucleotide sequence comprising apolynucleotide sequence selected from the group consisting of SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, a fragment of SEQ ID NO:2, a fragment ofSEQ ID NO:4, and a fragment of SEQ ID NO:6.

The invention further provides an expression vector containing at leasta fragment of the polynucleotide sequence encoding the polypeptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, a fragment of SEQ ID NO:1, afragment of SEQ ID NO:3, and a fragment of SEQ ID NO:5. In anotheraspect, the expression vector is contained within a host cell.

The invention also provides a method for producing a polypeptidecomprising the amino acid sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:5, a fragment of SEQ ID NO:1, a fragment of SEQ ID NO:3, or afragment of SEQ ID NO:5, the method comprising the steps of: (a)culturing the host cell containing an expression vector containing atleast a fragment of a polynucleotide sequence encoding APOP underconditions suitable for the expression of the polypeptide; and (b)recovering the polypeptide from the host cell culture.

The invention also provides a pharmaceutical composition comprising asubstantially purified APOP having the amino acid sequence of SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:5, a fragment of SEQ ID NO:1, a fragment ofSEQ ID NO:3, or a fragment of SEQ ID NO:5 in conjunction with a suitablepharmaceutical carrier.

The invention further includes a purified antibody which binds to apolypeptide comprising the amino acid sequence of SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:5, a fragment of SEQ ID NO:1, a fragment of SEQ ID NO:3,or a fragment of SEQ ID NO:5, as well as a purified agonist and apurified antagonist to the polypeptide.

The invention also provides a method for preventing or treating a cancercomprising administering to a subject in need of such treatment aneffective amount of a pharmaceutical composition comprising purifiedAPOP.

The invention also provides a method for preventing or treating adisorder associated with an increase in apoptosis comprisingadministering to a subject in need of such treatment an effective amountof a pharmaceutical composition comprising purified APOP.

The invention also provides a method for preventing or treating a cancercomprising administering to a subject in need of such treatment aneffective amount of an antagonist of APOP.

The invention also provides a method for preventing or treating aninflammation comprising administering to a subject in need of suchtreatment an effective amount of an antagonist of APOP.

The invention also provides a method for preventing or treating adisorder associated with an increase in apoptosis comprisingadministering to a subject in need of such treatment an effective amountof an antagonist of APOP.

The invention also provides a method for detecting a polynucleotideencoding APOP in a biological sample containing nucleic acids, themethod comprising the steps of: (a) hybridizing the complement of thepolynucleotide sequence encoding the polypeptide comprising SEQ ID NO:1,SEQ ID NO:3, SEQ ID NO:5, a fragment of SEQ ID NO:1, a fragment of SEQID NO:3, or a fragment of SEQ ID NO:5 to at least one of the nucleicacids of the biological sample, thereby forming a hybridization complex;and (b) detecting the hybridization complex, wherein the presence of thehybridization complex correlates with the presence of a polynucleotideencoding APOP in the biological sample. In one aspect, the nucleic acidsof the biological sample are amplified by the polymerase chain reactionprior to the hybridizing step.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A, 1B, and 1C show the amino acid sequence (SEQ ID NO:1) andnucleic acid sequence (SEQ ID NO:2) of APOP-1. The alignment wasproduced using MacDNASIS PRO™ software (Hitachi Software Engineering Co.Ltd. San Bruno, Calif.).

FIG. 2 shows the amino acid sequence alignments between APOP-1 (358637;SEQ ID NO: 1) and a human Bcl-2 binding component 6 (GI 1683637; SEQ IDNO:7), produced using the multisequence alignment program of DNASTAR™software (DNASTAR Inc, Madison Wis.).

FIGS. 3A and 3B show the hydrophobicity plots for APOP-1 (SEQ ID NO:1)and the human Bcl-2 binding component 6 (SEQ ID NO:7), respectively. Thepositive X axis reflects amino acid position, and the negative Y axis,hydrophobicity (MacDNASIS PRO software).

FIGS. 4A, 4B, 4C, 4D, 4E, and 4F show the amino acid sequence (SEQ IDNO:3) and nucleic acid sequence (SEQ ID NO:4) of APOP-2. The alignmentwas produced using MacDNASIS PRO™ software (Hitachi Software EngineeringCo. Ltd. San Bruno, Calif.).

FIGS. 5A and 5B show the amino acid sequence alignments between APOP-2(1352286; SEQ ID NO:3) and a human LRR protein, p37NB (GI 1236329; SEQID NO:8), produced using the multisequence alignment program of DNASTAR™software (DNASTAR Inc, Madison Wis.).

FIGS. 6A and 6B show the hydrophobicity plots for APOP-2 (SEQ ID NO:3)and p37NB (SEQ ID NO:8), respectively. The positive X axis reflectsamino acid position, and the negative Y axis, hydrophobicity (MacDNASISPRO software).

FIGS. 7A, 7B, 7C, 7D, 7E, 7F, and 7G show the amino acid sequence (SEQID NO:5) and nucleic acid sequence (SEQ ID NO:6) of APOP-3. Thealignment was produced using MacDNASIS PRO™ software (Hitachi SoftwareEngineering Co. Ltd. San Bruno, Calif.).

FIGS. 8A and 8B show the amino acid sequence alignments between APOP-3(815087; SEQ ID NO:5) and a mouse apoptosis inducible protein, MA-3 (GI1384078; SEQ ID NO:9), produced using the multisequence alignmentprogram of DNASTAR™ software (DNASTAR Inc, Madison Wis.).

FIGS. 9A and 9B show the hydrophobicity plots for APOP-3 (SEQ ID NO:5)and MA-3 (SEQ ID NO:9), respectively. The positive X axis reflects aminoacid position, and the negative Y axis, hydrophobicity (MacDNASIS PROsoftware).

DESCRIPTION OF THE INVENTION

Before the present proteins, nucleotide sequences, and methods aredescribed, it is understood that this invention is not limited to theparticular methodology, protocols, cell lines, vectors, and reagentsdescribed, as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms "a", "an", and "the" include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to "ahost cell" includes a plurality of such host cells, reference to the"antibody" is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methods,devices, and materials are now described. All publications mentionedherein are incorporated herein by reference for the purpose ofdescribing and disclosing the cell lines, vectors, and methodologieswhich are reported in the publications which might be used in connectionwith the invention. Nothing herein is to be construed as an admissionthat the invention is not entitled to antedate such disclosure by virtueof prior invention.

DEFINITIONS

APOP, as used herein, refers to the amino acid sequences ofsubstantially purified APOP obtained from any species, particularlymammalian, including bovine, ovine, porcine, murine, equine, andpreferably human, from any source whether natural, synthetic,semi-synthetic, or recombinant.

The term "agonist", as used herein, refers to a molecule which, whenbound to APOP, increases or prolongs the duration of the effect of APOP.Agonists may include proteins, nucleic acids, carbohydrates, or anyother molecules which bind to and modulate the effect of APOP.

An "allele" or "allelic sequence", as used herein, is an alternativeform of the gene encoding APOP. Alleles may result from at least onemutation in the nucleic acid sequence and may result in altered mRNAs orpolypeptides whose structure or function may or may not be altered. Anygiven natural or recombinant gene may have none, one, or many allelicforms. Common mutational changes which give rise to alleles aregenerally ascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

"Altered" nucleic acid sequences encoding APOP, as used herein, includethose with deletions, insertions, or substitutions of differentnucleotides resulting in a polynucleotide that encodes the same or afunctionally equivalent APOP. Included within this definition arepolymorphisms which may or may not be readily detectable using aparticular oligonucleotide probe of the polynucleotide encoding APOP,and improper or unexpected hybridization to alleles, with a locus otherthan the normal chromosomal locus for the polynucleotide sequenceencoding APOP. The encoded protein may also be "altered" and containdeletions, insertions, or substitutions of amino acid residues whichproduce a silent change and result in a functionally equivalent APOP.Deliberate amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues as long asthe biological or immunological activity of APOP is retained. Forexample, negatively charged amino acids may include aspartic acid andglutamic acid; positively charged amino acids may include lysine andarginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values may include leucine, isoleucine, andvaline, glycine and alanine, asparagine and glutamine, serine andthreonine, and phenylalanine and tyrosine.

"Amino acid sequence", as used herein, refers to an oligopeptide,peptide, polypeptide, or protein sequence, and fragment thereof, and tonaturally occurring or synthetic molecules. Fragments of APOP arepreferably about 5 to about 15 amino acids in length and retain thebiological activity or the immunological activity of APOP. Where "aminoacid sequence" is recited herein to refer to an amino acid sequence of anaturally occurring protein molecule, amino acid sequence, and liketerms, are not meant to limit the amino acid sequence to the complete,native amino acid sequence associated with the recited protein molecule.

"Amplification", as used herein, refers to the production of additionalcopies of a nucleic acid sequence and is generally carried out usingpolymerase chain reaction (PCR) technologies well known in the art(Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, a LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y.).

The term "antagonist", as used herein, refers to a molecule which, whenbound to APOP, decreases the amount or the duration of the effect of thebiological or immunological activity of APOP. Antagonists may includeproteins, nucleic acids, carbohydrates, antibodies or any othermolecules which decrease the effect of APOP.

As used herein, the term "antibody" refers to intact molecules as wellas fragments thereof, such as Fa, F(ab')₂, and Fv, which are capable ofbinding the epitopic determinant. Antibodies that bind APOP polypeptidescan be prepared using intact polypeptides or fragments containing smallpeptides of interest as the immunizing antigen. The polypeptide oroligopeptide used to immunize an animal can be derived from thetranslation of RNA or synthesized chemically and can be conjugated to acarrier protein, if desired. Commonly used carriers that are chemicallycoupled to peptides include bovine serum albumin and thyroglobulin,keyhole limpet hemocyanin. The coupled peptide is then used to immunizethe animal (e.g., a mouse, a rat, or a rabbit).

The term "antigenic determinant", as used herein, refers to thatfragment of a molecule (i.e., an epitope) that makes contact with aparticular antibody. When a protein or fragment of a protein is used toimmunize a host animal, numerous regions of the protein may induce theproduction of antibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the immunogen used to elicit theimmune response) for binding to an antibody.

The term "antisense", as used herein, refers to any compositioncontaining nucleotide sequences which are complementary to a specificDNA or RNA sequence. The term "antisense strand" is used in reference toa nucleic acid strand that is complementary to the "sense" strand.Antisense molecules include peptide nucleic acids and may be produced byany method including synthesis or transcription. Once introduced into acell, the complementary nucleotides combine with natural sequencesproduced by the cell to form duplexes and block either transcription ortranslation. The designation "negative" is sometimes used in referenceto the antisense strand, and "positive" is sometimes used in referenceto the sense strand.

The term "biologically active", as used herein, refers to a proteinhaving structural, regulatory, or biochemical functions of a naturallyoccurring molecule. Likewise, "immunologically active" refers to thecapability of the natural, recombinant, or synthetic APOP, or anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

The terms "complementary" or "complementarity", as used herein, refer tothe natural binding of polynucleotides under permissive salt andtemperature conditions by base-pairing. For example, the sequence"A-G-T" binds to the complementary sequence "T-C-A". Complementaritybetween two single-stranded molecules may be "partial", in which onlysome of the nucleic acids bind, or it may be complete when totalcomplementarity exists between the single stranded molecules. The degreeof complementarity between nucleic acid strands has significant effectson the efficiency and strength of hybridization between nucleic acidstrands. This is of particular importance in amplification reactions,which depend upon binding between nucleic acids strands and in thedesign and use of PNA molecules.

A "composition comprising a given polynucleotide sequence", as usedherein, refers broadly to any composition containing the givenpolynucleotide sequence. The composition may comprise a dry formulationor an aqueous solution. Compositions comprising polynucleotide sequencesencoding APOP (SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5) or fragmentsthereof (e.g., SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 and fragmentsthereof) may be employed as hybridization probes. The probes may bestored in freeze-dried form and may be associated with a stabilizingagent such as a carbohydrate. In hybridizations, the probe may bedeployed in an aqueous solution containing salts (e.g., NaCl),detergents (e.g., SDS) and other components (e.g., Denhardt's solution,dry milk, salmon sperm DNA, etc.).

"Consensus", as used herein, refers to a nucleic acid sequence which hasbeen resequenced to resolve uncalled bases, has been extended usingXL-PCR™ (Perkin Elmer, Norwalk, Conn.) in the 5' and/or the 3' directionand resequenced, or has been assembled from the overlapping sequences ofmore than one Incyte Clone using a computer program for fragmentassembly (e.g., GELVIEW™ Fragment Assembly system, GCG, Madison, Wis.).Some sequences have been both extended and assembled to produce theconsensus sequence.

The term "correlates with expression of a polynucleotide", as usedherein, indicates that the detection of the presence of ribonucleic acidthat is similar to SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6 by northernanalysis is indicative of the presence of mRNA encoding APOP in a sampleand thereby correlates with expression of the transcript from thepolynucleotide encoding the protein.

A "deletion", as used herein, refers to a change in the amino acid ornucleotide sequence and results in the absence of one or more amino acidresidues or nucleotides.

The term "derivative", as used herein, refers to the chemicalmodification of a nucleic acid encoding or complementary to APOP or theencoded APOP. Such modifications include, for example, replacement ofhydrogen by an alkyl, acyl, or amino group. A nucleic acid derivativeencodes a polypeptide which retains the biological or immunologicalfunction of the natural molecule. A derivative polypeptide is one whichis modified by glycosylation, pegylation, or any similar process whichretains the biological or immunological function of the polypeptide fromwhich it was derived.

The term "homology", as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology(i.e., identity). A partially complementary sequence that at leastpartially inhibits an identical sequence from hybridizing to a targetnucleic acid is referred to using the functional term "substantiallyhomologous." The inhibition of hybridization of the completelycomplementary sequence to the target sequence may be examined using ahybridization assay (Southern or northern blot, solution hybridizationand the like) under conditions of low stringency. A substantiallyhomologous sequence or hybridization probe will compete for and inhibitthe binding of a completely homologous sequence to the target sequenceunder conditions of low stringency. This is not to say that conditionsof low stringency are such that non-specific binding is permitted; lowstringency conditions require that the binding of two sequences to oneanother be a specific (i.e., selective) interaction. The absence ofnon-specific binding may be tested by the use of a second targetsequence which lacks even a partial degree of complementarity (e.g.,less than about 30% identity). In the absence of non-specific binding,the probe will not hybridize to the second non-complementary targetsequence.

Human artificial chromosomes (HACs) are linear microchromosomes whichmay contain DNA sequences of 10 K to 10M in size and contain all of theelements required for stable mitotic chromosome segregation andmaintenance (Harrington, J. J. et al. (1997) Nat Genet. 15:345-355).

The term "humanized antibody", as used herein, refers to antibodymolecules in which amino acids have been replaced in the non-antigenbinding regions in order to more closely resemble a human antibody,while still retaining the original binding ability.

The term "hybridization", as used herein, refers to any process by whicha strand of nucleic acid binds with a complementary strand through basepairing.

The term "hybridization complex", as used herein, refers to a complexformed between two nucleic acid sequences by virtue of the formation ofhydrogen bonds between complementary G and C bases and betweencomplementary A and T bases; these hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g., C₀ t or R₀ tanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized on a solid support (e.g.,paper, membranes, filters, chips, pins or glass slides, or any otherappropriate substrate to which cells or their nucleic acids have beenfixed).

An "insertion" or "addition", as used herein, refers to a change in anamino acid or nucleotide sequence resulting in the addition of one ormore amino acid residues or nucleotides, respectively, as compared tothe naturally occurring molecule.

"Microarray" refers to an array of distinct polynucleotides oroligonucleotides arranged on a substrate, such as paper, nylon or othertype of membrane, filter, chip, glass slide, or any other suitable solidsupport.

The term "modulate", as used herein, refers to a change in the activityof APOP. For example, modulation may cause an increase or a decrease inprotein activity, binding characteristics, or any other biological,functional or immunological properties of APOP.

"Nucleic acid sequence" as used herein refers to an oligonucleotide,nucleotide, or polynucleotide, and fragments thereof, and to DNA or RNAof genomic or synthetic origin which may be single- or double-stranded,and represent the sense or antisense strand. "Fragments" are thosenucleic acid sequences which are greater than 60 nucleotides than inlength, and most preferably includes fragments that are at least 100nucleotides or at least 1000 nucleotides, and at least 10,000nucleotides in length.

The term "oligonucleotide" refers to a nucleic acid sequence of at leastabout 6 nucleotides to about 60 nucleotides, preferably about 15 to 30nucleotides, and more preferably about 20 to 25 nucleotides, which canbe used in PCR amplification or a hybridization assay, or a microarray.As used herein, oligonucleotide is substantially equivalent to the terms"amplimers", "primers", "oligomers", and "probes", as commonly definedin the art.

"Peptide nucleic acid", PNA as used herein, refers to an antisensemolecule or anti-gene agent which comprises an oligonucleotide of atleast five nucleotides in length linked to a peptide backbone of aminoacid residues which ends in lysine. The terminal lysine conferssolubility to the composition. PNAs may be pegylated to extend theirlifespan in the cell where they preferentially bind complementary singlestranded DNA and RNA and stop transcript elongation (Nielsen, P. E. etal. (1993) Anticancer Drug Des. 8:53-63).

The term "portion", as used herein, with regard to a protein (as in "aportion of a given protein") refers to fragments of that protein. Thefragments may range in size from five amino acid residues to the entireamino acid sequence minus one amino acid. Thus, for example, a protein"comprising at least a portion of the amino acid sequence of SEQ IDNO:1" encompasses the full-length APOP-1 and fragments thereof.

The term "sample", as used herein, is used in its broadest sense. Abiological sample suspected of containing nucleic acid encoding APOP, orfragments thereof, or APOP itself may comprise a bodily fluid, extractfrom a cell, chromosome, organelle, or membrane isolated from a cell, acell, genomic DNA, RNA, or cDNA (in solution or bound to a solidsupport, a tissue, a tissue print, and the like).

The terms "specific binding" or "specifically binding", as used herein,refers to that interaction between a protein or peptide and an agonist,an antibody and an antagonist. The interaction is dependent upon thepresence of a particular structure (i.e., the antigenic determinant orepitope) of the protein recognized by the binding molecule. For example,if an antibody is specific for epitope "A", the presence of a proteincontaining epitope A (or free, unlabeled A) in a reaction containinglabeled "A" and the antibody will reduce the amount of labeled A boundto the antibody.

As used herein, the term "stringent conditions" refers to conditionswhich permit hybridization between polynucleotide sequences and theclaimed polynucleotide sequences. Suitably stringent conditions can bedefined by, for example, the concentrations of salt or formamide in theprehybridization and hybridization solutions, or by the hybridizationtemperature, and are well known in the art. In particular, stringencycan be increased by reducing the concentration of salt, increasing theconcentration of formamide, or raising the hybridization temperature.

For example, hybridization under high stringency conditions could occurin about 50% formamide at about 37° C. to 42° C. Hybridization couldoccur under reduced stringency conditions in about 35% to 25% formamideat about 30° C. to 35° C. In particular, hybridization could occur underhigh stringency conditions at 42° C. in 50% formamide, 5×SSPE, 0.3% SDS,and 200 μg/ml sheared and denatured salmon sperm DNA. Hybridizationcould occur under reduced stringency conditions as described above, butin 35% formamide at a reduced temperature of 35° C. The temperaturerange corresponding to a particular level of stringency can be furthernarrowed by calculating the purine to pyrimidine ratio of the nucleicacid of interest and adjusting the temperature accordingly. Variationson the above ranges and conditions are well known in the art.

The term "substantially purified", as used herein, refers to nucleic oramino acid sequences that are removed from their natural environment,isolated or separated, and are at least 60% free, preferably 75% free,and most preferably 90% free from other components with which they arenaturally associated.

A "substitution", as used herein, refers to the replacement of one ormore amino acids or nucleotides by different amino acids or nucleotides,respectively.

"Transformation", as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. It may occur undernatural or artificial conditions using various methods well known in theart. Transformation may rely on any known method for the insertion offoreign nucleic acid sequences into a prokaryotic or eukaryotic hostcell. The method is selected based on the type of host cell beingtransformed and may include, but is not limited to, viral infection,electroporation, heat shock, lipofection, and particle bombardment. Such"transformed" cells include stably transformed cells in which theinserted DNA is capable of replication either as an autonomouslyreplicating plasmid or as part of the host chromosome. They also includecells which transiently express the inserted DNA or RNA for limitedperiods of time.

A "variant" of APOP, as used herein, refers to an amino acid sequencethat is altered by one or more amino acids. The variant may have"conservative" changes, wherein a substituted amino acid has similarstructural or chemical properties, e.g., replacement of leucine withisoleucine. More rarely, a variant may have "nonconservative" changes,e.g., replacement of a glycine with a tryptophan. Analogous minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, DNASTAR software.

THE INVENTION

The invention is based on the discovery of three new human proteinsassociated with cell proliferation (hereinafter collectively referred toas "APOP"), the polynucleotides encoding APOP, and the use of thesecompositions for the diagnosis, prevention, or treatment of disordersassociated with abnormal cell proliferation and apoptosis.

Nucleic acids encoding the APOP-1 of the present invention were firstidentified in Incyte Clone 358673 from a synovial cDNA library(SYNORAB01) using a computer search for amino acid sequence alignments.A consensus sequence, SEQ ID NO:2, was derived from the followingoverlapping and/or extended nucleic acid sequences: Incyte Clones 358673(SYNORAB01) and 1663788 (BRSTNOT09).

In one embodiment, the invention encompasses a polypeptide, APOP-1,comprising the amino acid sequence of SEQ ID NO:1, as shown in FIGS. 1A,1B, and 1C. APOP-1 is 168 amino acids in length. APOP-1 has onepotential cAMP- and cGMP-dependent protein kinase phosphorylation siteencompassing residues R115-S118; four potential casein kinase IIphosphorylation sites encompassing residues S10-E13, S16-E19, T80-D83,and S153-D156; and two potential protein kinase C phosphorylation sitesencompassing residues S34-K36 and S124-K126. As shown in FIGS. 2A and2B, APOP-1 has chemical and structural homology with a human Bcl-2binding component 6 (GI 1683637; SEQ ID NO:7). In particular, APOP-1 andBcl-2 binding component 6 share 85% sequence homology. As illustrated byFIGS. 3A and 3B, APOP-1 and Bcl-2 binding component 6 have rathersimilar hydrophobicity plots. Northern analysis shows the expression ofAPOP-1 in various cDNA libraries, at least 68% of which are immortalizedor cancerous, at least 17% of which involve immune response, and atleast 12% of which are expressed in fetal/infant tissues or organs.

Nucleic acids encoding the APOP-2 of the present invention were firstidentified in Incyte Clone 1352286 from a myoma tissue cDNA library(LATRTUT02) using a computer search for amino acid sequence alignments.A consensus sequence, SEQ ID NO:4, was derived from the followingoverlapping and/or extended nucleic acid sequences: Incyte Clones1901255 (BLADTUT06), 3279966 and 3281759 (STOMFET02), 3141791(SMCCNOT02), 3133726 (SMCCNOT01), 1349544, 1407110 and 1352286(LATRTUT02), and 1357338 (LUNGNOT09).

In one embodiment, the invention encompasses a polypeptide, APOP-2,comprising the amino acid sequence of SEQ ID NO:3, as shown in FIGS. 4A,4B, and 4C. APOP-2 is 440 amino acids in length. Similar to p37NB (GI1236329, SEQ ID NO:8), a putative LRR protein, APOP-2 has several LRRmotifs which are potentially involved in protein-protein interaction.APOP-2 also has one potential amidation site encompassing residuesG37-R40, two potential N-glycosylation sites encompassing residuesN297-S300 and N324-L327; three potential cAMP- and cGMP-dependentprotein kinase phosphorylation sites encompassing residues R19-S22,R39-S42, and K430-S433; six potential casein kinase II phosphorylationsites encompassing residues S118-E121, S194-E197, T315-E318, T379-E382,S394-E397, and T415-D418; and four potential protein kinase Cphosphorylation sites encompassing residues S25-R27, T95-K97, S226-R228,and T428-K430. As shown in FIG. 5, APOP-2 has chemical and structuralhomology with a human LRR protein, p37NB (GI 1236329; SEQ ID NO:8). Inparticular, APOP-2 and p37NB share 74% sequence homology. As illustratedby FIGS. 6A and 6B, APOP-2 and p37NB have rather similar hydrophobicityplots. Northern analysis shows the expression of APOP-2 in various cDNAlibraries, at least 27% of which are immortalized or cancerous, at least15% of which involve immune response, and at least 50% of which areexpressed in fetal/infant tissues or organs.

Nucleic acids encoding the APOP-3 of the present invention were firstidentified in Incyte Clone 815087 from an ovarian tumor tissue cDNAlibrary (OVARTUT01) using a computer search for amino acid sequencealignments. A consensus sequence, SEQ ID NO:6, was derived from thefollowing overlapping and/or extended nucleic acid sequences: IncyteClones 1697887 (BLADTUT05), 140813 (TLYMNOR01), 1291763 (PGANNOT03),2741788 (BRSTTUT14), 2236154 (PANCTUT02), 1610527 (COLNTUT06), 2518507(BRAITUT21), 815087 (OVARTUT01), and sequence SAEA00394.

In one embodiment, the invention encompasses a polypeptide, APOP-3,comprising the amino acid sequence of SEQ ID NO:5, as shown in FIGS. 7A,7B, 7C, 7D, 7E, 7F, and 7G. APOP-3 is 469 amino acids in length. APOP-3has two potential N-glycosylation sites encompassing residues N18-D21and N66-R69; two potential cAMP- and cGMP-dependent protein kinasephosphorylation sites encompassing residues R64-S67 and R310-S313; 13potential casein kinase II phosphorylation sites encompassing residuesS25-E28, S49-E52, S67-D70, S76-D79, S80-D83, S214-E217, S232-D235,T234-E237, T277-D280, S345-E348, S374-E377, S424-E427, and S457-D460;nine potential protein kinase C phosphorylation sites encompassingresidues S67-R69, S71-R73, S94-K96, S106-K108, S214-R216, T219-K221,T254-R256, S281-K283, and T379-K381; one potential cell attachmentsequence encompassing residues R73-D75; and one potential tyrosinekinase phosphorylation site encompassing residues K402-Y409. As shown inFIG. 8, APOP-3 has chemical and structural homology with a mouseapoptosis-associated protein, MA-3 (GI 1384078; SEQ ID NO:9). Inparticular, APOP-3 and MA-3 share 96% sequence homology. As illustratedby FIGS. 9A and 9B, APOP-3 and MA-3 have rather similar hydrophobicityplots. Northern analysis shows the expression of APOP-3 in various cDNAlibraries, at least 48% of which are immortalized or cancerous, and atleast 30% of which involve immune response, and at least 8% of which areexpressed in fetal/infant tissues or organs.

The invention also encompasses APOP variants. A preferred APOP variantis one which has at least about 80%, more preferably at least about 90%,and most preferably at least about 95% amino acid sequence identity tothe APOP amino acid sequence, and which contains at least onebiological,immunological or other functional characteristic or activity of APOP. Amost preferred APOP variant is one having at least 95% amino acidsequence which encodes APOP.

The invention also encompasses polynucleotides which encode APOP.Accordingly, any nucleic acid sequence which encodes the amino acidsequence of APOP can be used to produce recombinant molecules whichexpress APOP. In a particular embodiment, the invention encompasses apolynucleotide sequence comprising the sequence of SEQ ID NO:2, asshown, in FIGS. 1A, 1B, and 1C, which encodes an APOP. In a furtherembodiment, the invention encompasses a polynucleotide sequencecomprising the sequence of SEQ ID NO:4, as shown in FIGS. 4A, 4B, 4C,4D, and 4F, which encodes an APOP. Still further, the inventionencompasses a polynucleotide sequence comprising the sequence of SEQ IDNO:6, as shown in FIGS. 7A, 7B, 7C, 7D, 7E, 7F and 7G, which encodes anAPOP.

The invention also encompasses a variant of a polynucleotide sequenceencoding APOP. In particular, such a variant polynucleotide sequencewill have at least about 80%, more preferably at least about 90%, andmost preferably at least about 95% polynucleotide sequence identity tothe polynucleotide sequence encoding APOP. A particular aspect of theinvention encompasses a variant of SEQ ID NO:2, which has at least about80%, more preferably at least about 90%, and most preferably at leastabout 95% polynucleotide sequence identity to SEQ ID NO:2. The inventionfurther encompasses a polynucleotide variant of SEQ ID NO:4 having atleast about 80%, more preferably at least about 90%, and most preferablyat least about 95% polynucleotide sequence identity to SEQ ID NO:4.Still further, the invention encompasses a polynucleotide variant of SEQID NO:6 having at least about 80%, more preferably at least about 90%,and most preferably at least about 95% polynucleotide sequence identityto SEQ ID NO:6. Any one of the polynucleotide variants described abovecan encode an amino acid sequence which contains at least onebiological, immunological or other functional characteristic or activityof APOP.

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of nucleotide sequencesencoding APOP, some bearing minimal homology to the nucleotide sequencesof any known and naturally occurring gene, may be produced. Thus, theinvention contemplates each and every possible variation of nucleotidesequence that could be made by selecting combinations based on possiblecodon choices. These combinations are made in accordance with thestandard triplet genetic code as applied to the nucleotide sequence ofnaturally occurring APOP, and all such variations are to be consideredas being specifically disclosed.

Although nucleotide sequences which encode APOP and its variants arepreferably capable of hybridizing to the nucleotide sequence of thenaturally occurring APOP under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding APOP or its derivatives possessing a substantially differentcodon usage. Codons may be selected to increase the rate at whichexpression of the peptide occurs in a particular prokaryotic oreukaryotic host in accordance with the frequency with which particularcodons are utilized by the host. Other reasons for substantiallyaltering the nucleotide sequence encoding APOP and its derivativeswithout altering the encoded amino acid sequences include the productionof RNA transcripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringsequence.

The invention also encompasses production of DNA sequences, or fragmentsthereof, which encode APOP and its derivatives, entirely by syntheticchemistry. After production, the synthetic sequence may be inserted intoany of the many available expression vectors and cell systems usingreagents that are well known in the art. Moreover, synthetic chemistrymay be used to introduce mutations into a sequence encoding APOP or anyfragment thereof.

Also encompassed by the invention are polynucleotide sequences that arecapable of hybridizing to the claimed nucleotide sequences, and, inparticular, those shown in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6,under various conditions of stringency as taught in Wahl, G. M. and S.L. Berger (1987; Methods Enzymol. 152:399-407) and Kimmel, A. R. (1987;Methods Enzymol. 152:507-511).

Methods for DNA sequencing which are well known and generally availablein the art and may be used to practice any of the embodiments of theinvention. The methods may employ such enzymes as the Klenow fragment ofDNA polymerase I, Sequenase® (US Biochemical Corp, Cleveland, Ohio), Taqpolymerase (Perkin Elmer), thermostable T7 polymerase (Amersham,Chicago, Ill.), or combinations of polymerases and proofreadingexonucleases such as those found in the ELONGASE Amplification Systemmarketed by GIBCO/BRL (Gaithersburg, Md.). Preferably, the process isautomated with machines such as the Hamilton Micro Lab 2200 (Hamilton,Reno, Nev.), Peltier Thermal Cycler (PTC200; MJ Research, Watertown,Mass.) and the ABI Catalyst and 373 and 377 DNA Sequencers (PerkinElmer).

The nucleic acid sequences encoding APOP may be extended utilizing apartial nucleotide sequence and employing various methods known in theart to detect upstream sequences such as promoters and regulatoryelements. For example, one method which may be employed,"restriction-site" PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus (Sarkar, G. (1993) PCR MethodsApplic. 2:318-322). In particular, genomic DNA is first amplified in thepresence of primer to a linker sequence and a primer specific to theknown region. The amplified sequences are then subjected to a secondround of PCR with the same linker primer and another specific primerinternal to the first one. Products of each round of PCR are transcribedwith an appropriate RNA polymerase and sequenced using reversetranscriptase.

Inverse PCR may also be used to amplify or extend sequences usingdivergent primers based on a known region (Triglia, T. et al. (1988)Nucleic Acids Res. 16:8186). The primers may be designed usingcommercially available software such as OLIGO 4.06 Primer Analysissoftware (National Biosciences Inc., Plymouth, Minn.), or anotherappropriate program, to be 22-30 nucleotides in length, to have a GCcontent of 50% or more, and to anneal to the target sequence attemperatures about 68°-72° C. The method uses several restrictionenzymes to generate a suitable fragment in the known region of a gene.The fragment is then circularized by intramolecular ligation and used asa PCR template.

Another method which may be used is capture PCR which involves PCRamplification of DNA fragments adjacent to a known sequence in human andyeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCRMethods Applic. 1:111-119). In this method, multiple restriction enzymedigestions and ligations may also be used to place an engineereddouble-stranded sequence into an unknown fragment of the DNA moleculebefore performing PCR.

Another method which may be used to retrieve unknown sequences is thatof Parker, J. D. et al. (1991; Nucleic Acids Res. 19:3055-3060).Additionally, one may use PCR, nested primers, and PromoterFinder™libraries to walk genomic DNA (Clontech, Palo Alto, Calif.). Thisprocess avoids the need to screen libraries and is useful in findingintron/exon junctions.

When screening for full-length cDNAs, it is preferable to use librariesthat have been size-selected to include larger cDNAs. Also,random-primed libraries are preferable, in that they will contain moresequences which contain the 5' regions of genes. Use of a randomlyprimed library may be especially preferable for situations in which anoligo d(T) library does not yield a full-length cDNA. Genomic librariesmay be useful for extension of sequence into 5' non-transcribedregulatory regions.

Capillary electrophoresis systems which are commercially available maybe used to analyze the size or confirm the nucleotide sequence ofsequencing or PCR products. In particular, capillary sequencing mayemploy flowable polymers for electrophoretic separation, four differentfluorescent dyes (one for each nucleotide) which are laser activated,and detection of the emitted wavelengths by a charge coupled devicecamera. Output/light intensity may be converted to electrical signalusing appropriate software (e.g. Genotyper™ and Sequence Navigator™,Perkin Elmer) and the entire process from loading of samples to computeranalysis and electronic data display may be computer controlled.Capillary electrophoresis is especially preferable for the sequencing ofsmall pieces of DNA which might be present in limited amounts in aparticular sample.

In another embodiment of the invention, polynucleotide sequences orfragments thereof which encode APOP may be used in recombinant DNAmolecules to direct expression of APOP, fragments or functionalequivalents thereof, in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced, and these sequences may be used to clone and expressAPOP.

As will be understood by those of skill in the art, it may beadvantageous to produce APOP-encoding nucleotide sequences possessingnon-naturally occurring codons. For example, codons preferred by aparticular prokaryotic or eukaryotic host can be selected to increasethe rate of protein expression or to produce an RNA transcript havingdesirable properties, such as a half-life which is longer than that of atranscript generated from the naturally occurring sequence.

The nucleotide sequences of the present invention can be engineeredusing methods generally known in the art in order to alter APOP encodingsequences for a variety of reasons, including but not limited to,alterations which modify the cloning, processing, and/or expression ofthe gene product. DNA shuffling by random fragmentation and PCRreassembly of gene fragments and synthetic oligonucleotides may be usedto engineer the nucleotide sequences. For example, site-directedmutagenesis may be used to insert new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, introduce mutations, and so forth.

In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding APOP may be ligated to aheterologous sequence to encode a fusion protein. For example, to screenpeptide libraries for inhibitors of APOP activity, it may be useful toencode a chimeric APOP protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between the APOP encoding sequence and theheterologous protein sequence, so that APOP may be cleaved and purifiedaway from the heterologous moiety.

In another embodiment, sequences encoding APOP may be synthesized, inwhole or in part, using chemical methods well known in the art (seeCaruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223,Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232).Alternatively, the protein itself may be produced using chemical methodsto synthesize the amino acid sequence of APOP, or a fragment thereof.For example, peptide synthesis can be performed using varioussolid-phase techniques (Roberge, J. Y. et al. (1995) Science269:202-204) and automated synthesis may be achieved, for example, usingthe ABI 431A Peptide Synthesizer (Perkin Elmer).

The newly synthesized peptide may be substantially purified bypreparative high performance liquid chromatography (e.g., Creighton, T.(1983) Proteins, Structures and Molecular Principles, WH Freeman andCo., New York, N.Y.). The composition of the synthetic peptides may beconfirmed by amino acid analysis or sequencing (e.g., the Edmandegradation procedure; Creighton, supra). Additionally, the amino acidsequence of APOP, or any part thereof, may be altered during directsynthesis and/or combined using chemical methods with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

In order to express a biologically active APOP, the nucleotide sequencesencoding APOP or functional equivalents, may be inserted intoappropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence.

Methods which are well known to those skilled in the art may be used toconstruct expression vectors containing sequences encoding APOP andappropriate transcriptional and translational control elements. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. Such techniques aredescribed in Sambrook, J. et al. (1989) Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. etal. (1989) Current Protocols in Molecular Biology, John Wiley & Sons,New York, N.Y.

A variety of expression vector/host systems may be utilized to containand express sequences encoding APOP. These include, but are not limitedto, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems. Theinvention is not limited by the host cell employed.

The "control elements" or "regulatory sequences" are thosenon-translated regions of the vector--enhancers, promoters, 5' and 3'untranslated regions--which interact with host cellular proteins tocarry out transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the Bluescript® phagemid (Stratagene,LaJolla, Calif.) or pSport1™ plasmid (Gibco BRL) and the like may beused. The baculovirus polyhedrin promoter may be used in insect cells.Promoters or enhancers derived from the genomes of plant cells (e.g.,heat shock, RUBISCO; and storage protein genes) or from plant viruses(e.g., viral promoters or leader sequences) may be cloned into thevector. In mammalian cell systems, promoters from mammalian genes orfrom mammalian viruses are preferable. If it is necessary to generate acell line that contains multiple copies of the sequence encoding APOP,vectors based on SV40 or EBV may be used with an appropriate selectablemarker.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for APOP. For example, when largequantities of APOP are needed for the induction of antibodies, vectorswhich direct high level expression of fusion proteins that are readilypurified may be used. Such vectors include, but are not limited to, themultifunctional E. coli cloning and expression vectors such asBluescript® (Stratagene), in which the sequence encoding APOP may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of β-galactosidase so that a hybridprotein is produced; pIN vectors (Van Heeke, G. and S. M. Schuster(1989) J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors(Promega, Madison, Wis.) may also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. Proteins made in suchsystems may be designed to include heparin, thrombin, or factor XAprotease cleavage sites so that the cloned polypeptide of interest canbe released from the GST moiety at will.

In the yeast, Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase, and PGH may be used. For reviews, see Ausubel et al. (supra)and Grant et al. (1987) Methods Enzymol. 153:516-544.

In cases where plant expression vectors are used, the expression ofsequences encoding APOP may be driven by any of a number of promoters.For example, viral promoters such as the 35S and 19S promoters of CaMVmay be used alone or in combination with the omega leader sequence fromTMV (Takamatsu, N. (1987) EMBO J. 6:307-311). Alternatively, plantpromoters such as the small subunit of RUBISCO or heat shock promotersmay be used (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R.et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) ResultsProbl. Cell Differ. 17:85-105). These constructs can be introduced intoplant cells by direct DNA transformation or pathogen-mediatedtransfection. Such techniques are described in a number of generallyavailable reviews (see, for example, Hobbs, S. or Murry, L. E. in McGrawHill Yearbook of Science and Technology (1992) McGraw Hill, New York,N.Y.; pp. 191-196).

An insect system may also be used to express APOP. For example, in onesuch system, Autographa californica nuclear polyhedrosis virus (AcNPV)is used as a vector to express foreign genes in Spodoptera frugiperdacells or in Trichoplusia larvae. The sequences encoding APOP may becloned into a non-essential region of the virus, such as the polyhedringene, and placed under control of the polyhedrin promoter. Successfulinsertion of APOP will render the polyhedrin gene inactive and producerecombinant virus lacking coat protein. The recombinant viruses may thenbe used to infect, for example, S. frugiperda cells or Trichoplusialarvae in which APOP may be expressed (Engelhard, E. K. et al. (1994)Proc. Nat. Acad. Sci. 91:3224-3227).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, sequences encoding APOP may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain a viable virus which iscapable of expressing APOP in infected host cells (Logan, J. and Shenk,T. (1984) Proc. Natl. Acad. Sci. 81:3655-3659). In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells.

Human artificial chromosomes (HACs) may also be employed to deliverlarger fragments of DNA than can be contained and expressed in aplasmid. HACs of 6 to 10M are constructed and delivered via conventionaldelivery methods (liposomes, polycationic amino polymers, or vesicles)for therapeutic purposes.

Specific initiation signals may also be used to achieve more efficienttranslation of sequences encoding APOP. Such signals include the ATGinitiation codon and adjacent sequences. In cases where sequencesencoding APOP, its initiation codon, and upstream sequences are insertedinto the appropriate expression vector, no additional transcriptional ortranslational control signals may be needed. However, in cases whereonly coding sequence, or a fragment thereof, is inserted, exogenoustranslational control signals including the ATG initiation codon shouldbe provided. Furthermore, the initiation codon should be in the correctreading frame to ensure translation of the entire insert. Exogenoustranslational elements and initiation codons may be of various origins,both natural and synthetic. The efficiency of expression may be enhancedby the inclusion of enhancers which are appropriate for the particularcell system which is used, such as those described in the literature(Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162).

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a "prepro" form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells which have specific cellular machineryand characteristic mechanisms for post-translational activities (e.g.,CHO, HeLa, MDCK, HEK293, and WI38), are available from the American TypeCulture Collection (ATCC; Bethesda, Md.) and may be chosen to ensure thecorrect modification and processing of the foreign protein.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressAPOP may be transformed using expression vectors which may contain viralorigins of replication and/or endogenous expression elements and aselectable marker gene on the same or on a separate vector. Followingthe introduction of the vector, cells may be allowed to grow for 1-2days in an enriched media before they are switched to selective media.The purpose of the selectable marker is to confer resistance toselection, and its presence allows growth and recovery of cells whichsuccessfully express the introduced sequences. Resistant clones ofstably transformed cells may be proliferated using tissue culturetechniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler, M. et al. (1977) Cell 11:223-32) and adeninephosphoribosyltransferase (Lowy, I. et al. (1980) Cell 22:817-23) geneswhich can be employed in tk⁻ or aprt⁻ cells, respectively. Also,antimetabolite, antibiotic or herbicide resistance can be used as thebasis for selection; for example, dhfr which confers resistance tomethotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.77:3567-70); npt, which confers resistance to the aminoglycosidesneomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol.150:1-14) and als or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hartman,S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51).Recently, the use of visible markers has gained popularity with suchmarkers as anthocyanins, β glucuronidase and its substrate GUS, andluciferase and its substrate luciferin, being widely used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector system(Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-131).

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, its presence and expression mayneed to be confirmed. For example, if the sequence encoding APOP isinserted within a marker gene sequence, transformed cells containingsequences encoding APOP can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with asequence encoding APOP under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the tandem gene as well.

Alternatively, host cells which contain the nucleic acid sequenceencoding APOP and express APOP may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridizations and proteinbioassay or immunoassay techniques which include membrane, solution, orchip based technologies for the detection and/or quantification ofnucleic acid or protein.

The presence of polynucleotide sequences encoding APOP can be detectedby DNA-DNA or DNA-RNA hybridization or amplification using probes orfragments or fragments of polynucleotides encoding APOP. Nucleic acidamplification based assays involve the use of oligonucleotides oroligomers based on the sequences encoding APOP to detect transformantscontaining DNA or RNA encoding APOP.

A variety of protocols for detecting and measuring the expression ofAPOP, using either polyclonal or monoclonal antibodies specific for theprotein are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescenceactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson APOP is preferred, but a competitive binding assay may be employed.These and other assays are described, among other places, in Hampton, R.et al. (1990; Scrological Methods, a Laboratory Manual, APS Press, StPaul, Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.158:1211-1216).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides encoding APOP includeoligolabeling, nick translation, end-labeling or PCR amplification usinga labeled nucleotide. Alternatively, the sequences encoding APOP, or anyfragments thereof may be cloned into a vector for the production of anmRNA probe. Such vectors are known in the art, are commerciallyavailable, and may be used to synthesize RNA probes in vitro by additionof an appropriate RNA polymerase such as T7, T3, or SP6 and labelednucleotides. These procedures may be conducted using a variety ofcommercially available kits (Pharmacia & Upjohn, (Kalamazoo, Mich.);Promega (Madison Wis.); and U.S. Biochemical Corp., Cleveland, Ohio).Suitable reporter molecules or labels, which may be used for ease ofdetection, include radionuclides, enzymes, fluorescent,chemiluminescent, or chromogenic agents as well as substrates,cofactors, inhibitors, magnetic particles, and the like.

Host cells transformed with nucleotide sequences encoding APOP may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a transformedcell may be secreted or contained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides which encodeAPOP may be designed to contain signal sequences which direct secretionof APOP through a prokaryotic or eukaryotic cell membrane. Otherconstructions may be used to join sequences encoding APOP to nucleotidesequence encoding a polypeptide domain which will facilitatepurification of soluble proteins. Such purification facilitating domainsinclude, but are not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp., Seattle, Wash.). The inclusion ofcleavable linker sequences such as those specific for Factor XA orenterokinase (Invitrogen, San Diego, Calif.) between the purificationdomain and APOP may be used to facilitate purification. One suchexpression vector provides for expression of a fusion protein containingAPOP and a nucleic acid encoding 6 histidine residues preceding athioredoxin or an enterokinase cleavage site. The histidine residuesfacilitate purification on IMAC (immobilized metal ion affinitychromatography as described in Porath, J. et al. (1992, Prot. Exp.Purif. 3: 263-281) while the enterokinase cleavage site provides a meansfor purifying APOP from the fusion protein. A discussion of vectorswhich contain fusion proteins is provided in Kroll, D. J. et al. (1993;DNA Cell Biol. 12:441-453).

In addition to recombinant production, fragments of APOP may be producedby direct peptide synthesis using solid-phase techniques (Merrifield J.(1963) J. Am. Chem. Soc. 85:2149-2154). Protein synthesis may beperformed using manual techniques or by automation. Automated synthesismay be achieved, for example, using Applied Biosystems 431A PeptideSynthesizer (Perkin Elmer). Various fragments of APOP may be chemicallysynthesized separately and combined using chemical methods to producethe full length molecule.

THERAPEUTICS

Chemical and structural homology exists between APOP-1 and a human Bcl-2binding component 6 (GI 1683637; SEQ ID NO:7); between APOP-2 and ahuman LRR protein, p37NB (GI 1236329; SEQ ID NO:8); and between APOP-3and a mouse apoptosis inducible protein, MA-3 (GI 1384078; SEQ ID NO:9).Northern analysis of APOP (SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5)expression suggests a close association with cell proliferation,inflammation, and fetal/infant development. Therapeutic uses for allthree polypeptides are described collectively below.

In cancers where APOP inhibits cell prolferation, it is desirable toincrease the expression of APOP. Therefore, in one embodiment, APOP, ora fragment or a derivative thereof, may be administered to a subject toprevent or treat cancer including, but not limited to, adenocarcinoma,leukemia, lymphoma, melanoma, myecloma, sarcoma, and teratocarcinoma,and, in particular, cancers of the adrenal gland, bladder, bone, bonemarrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinaltract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid,penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid,and uterus.

In another embodiment, a pharmaceutical composition comprising APOP maybe administered to a subject to prevent or treat a cancer including, butnot limited to, those listed above.

In still another embodiment, an agonist which is specific for APOP maybe administered to prevent or treat a cancer including, but not limitedto, those listed above.

In a further embodiment, a vector capable of expressing APOP, or afragment or a derivative thereof, may be used to prevent or treat acancer including, but not limited to, those listed above.

In disorders associated with an increase in apoptosis where APOPinhibits apoptosis, it is desirable to increase the expression of APOP.Therefore, in one embodiment, APOP or a fragment or derivative thereofmay be administered to a subject to prevent or treat a disorderassociated with an increase in apoptosis. Such disorders include, butare not limited to, AIDS and other infectious or geneticimmunodeficiencies, neurodegenerative diseases such as Alzheimer'sdisease, Parkinson's disease, amyotrophic lateral sclerosis, retinitispigmentosa, and cerebellar degeneration, myelodysplastic syndromes suchas aplastic anemia, ischemic injuries such as myocardial infarction,stroke, and reperfusion injury, toxin-induced diseases such asalcohol-induced liver damage, cirrhosis, and lathyrism, wasting diseasessuch as cachexia, viral infections such as those caused by hepatitis Band C, and osteoporosis.

In another embodiment, a pharmaceutical composition comprising APOP maybe administered to a subject to prevent or treat a disorder associatedwith increased apoptosis including, but not limited to, those listedabove.

In still another embodiment, an agonist which is specific for APOP maybe administered to prevent or treat a disorder associated with increasedapoptosis including, but not limited to, those listed above.

In a further embodiment, a vector capable of expressing APOP, or afragment or a derivative thereof, may be used to prevent or treat adisorder associated with increased apoptosis including, but not limitedto, those listed above.

In cancers where APOP promotes cell prolferation, it is desirable todecrease its activity. Therefore, in one embodiment, an antagonist ofAPOP, or a fragment or a derivative thereof, may be administered to asubject to prevent or treat cancer including, but not limited to,adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, andteratocarcinoma, and, in particular, cancers of the adrenal gland,bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus. In one aspect, an antibodyspecific for APOP may be used directly as an antagonist, or indirectlyas a targeting or delivery mechanism for bringing a pharmaceutical agentto cells or tissue which express APOP.

In another embodiment, a vector expressing the complement of thepolynucleotide encoding APOP, or a fragment or a derivative thereof, maybe administered to a subject to prevent or treat a disorder associatedwith cell proliferation including, but not limited to, the types ofcancer listed above.

In inflammation where APOP promotes cell proliferation, it is desirableto decrease its activity. Therefore, in one embodiment, an antagonist ofAPOP, or a fragment or a derivative thereof, may be administered to asubject to prevent or treat an inflammation. Disorders associated withinflammation include, but are not limited to, Addison's disease, adultrespiratory distress syndrome, allergies, anemia, asthma,atherosclerosis, bronchitis, cholecystitis, Crohn's disease, ulcerativecolitis, atopic dermatitis, dermatomyositis, diabetes mellitus,emphysema, atrophic gastritis, glomerulonephritis, gout, Graves'disease, hypereosinophilia, irritable bowel syndrome, lupuserythematosus, multiple sclerosis, myasthenia gravis, myocardial orpericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,polymyositis, rheumatoid arthritis, scleroderma, Sjogren's syndrome, andautoimmune thyroiditis; complications of cancer, hemodialysis,extracorporeal circulation; viral, bacterial, fungal, parasitic,protozoal, and helminthic infections and trauma. In one aspect, anantibody specific for APOP may be used directly as an antagonist, orindirectly as a targeting or delivery mechanism for bringing apharmaceutical agent to cells or tissue which express APOP.

In another embodiment, a vector expressing the complement of thepolynucleotide encoding APOP, or a fragment or a derivative thereof, maybe administered to a subject to prevent or treat an inflammationassociated with any disorder including, but not limited to, those listedabove.

In disorders associated with an increase in apoptosis where APOPstimulates apoptosis, it is desirable to decrease its activity.Therefore, in one enbodiment, an antagonist of APOP or a fragment orderivative thereof may be added to cells to stimulate cellproliferation. In particular, APOP may be added to a cell or cells invivo using delivery mechanisms such as liposomes, viral based vectors,or electroinjection for the purpose of promoting regeneration or celldifferentiation of the cell or cells. In addition, APOP may be added toa cell, cell line, tissue or organ culture in vitro or ex vivo tostimulate cell proliferation for use in heterologous or autologoustransplantation. In some cases, the cell will have been selected for itsability to fight an infection or a cancer or to correct a genetic defectin a disease such as sickle cell anemia, β thalassemia, cystic fibrosis,or Huntington's chorea. In one aspect, an antibody specific for APOP maybe used directly as an antagonist, or indirectly as a targeting ordelivery mechanism for bringing a pharmaceutical agent to cells ortissue which express APOP.

In another further embodiment, a vector expressing the complement of thepolynucleotide encoding APOP, or a fragment or a derivative thereof, maybe may be added to cells to stimulate cell proliferation, as describedabove.

In other embodiments, any of the proteins, antagonists, antibodies,agonists, complementary sequences, or vectors of the invention may beadministered in combination with other appropriate therapeutic agents.Selection of the appropriate agents for use in combination therapy maybe made by one of ordinary skill in the art, according to conventionalpharmaceutical principles. The combination of therapeutic agents may actsynergistically to effect the treatment or prevention of the variousdisorders described above. Using this approach, one may be able toachieve therapeutic efficacy with lower dosages of each agent, thusreducing the potential for adverse side effects.

An antagonist of APOP may be produced using methods which are generallyknown in the art. In particular, purified APOP may be used to produceantibodies or to screen libraries of pharmaceutical agents to identifythose which specifically bind APOP.

Antibodies to APOP may be generated using methods that are well known inthe art. Such antibodies may include, but are not limited to,polyclonal, monoclonal, chimeric, single chain, Fab fragments, andfragments produced by a Fab expression library. Neutralizing antibodies,(i.e., those which inhibit dimer formation) are especially preferred fortherapeutic use.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others, may be immunized by injectionwith APOP or any fragment or oligopeptide thereof which has immunogenicproperties. Depending on the host species, various adjuvants may be usedto increase immunological response. Such adjuvants include, but are notlimited to, Freund's, mineral gels such as aluminum hydroxide, andsurface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol. Among adjuvants used in humans, BCG (bacilliCalmette-Guerin) and Corynebacterium parvum are especially preferable.

It is preferred that the oligopeptides, peptides, or fragments used toinduce antibodies to APOP have an amino acid sequence consisting of atleast five amino acids and more preferably at least 10 amino acids. Itis also preferable that they are identical to a portion of the aminoacid sequence of the natural protein, and they may contain the entireamino acid sequence of a small, naturally occurring molecule. Shortstretches of APOP amino acids may be fused with those of another proteinsuch as keyhole limpet hemocyanin and antibody produced against thechimeric molecule.

Monoclonal antibodies to APOP may be prepared using any technique whichprovides for the production of antibody molecules by continuous celllines in culture. These include, but are not limited to, the hybridomatechnique, the human B-cell hybridoma technique, and the EBV-hybridomatechnique (Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. etal. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. et al. (1983) Proc.Natl. Acad. Sci. 80:2026-2030; Cole, S. P. et al. (1984) Mol. Cell Biol.62:109-120).

In addition, techniques developed for the production of "chimericantibodies", the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison, S. L. et al. (1984) Proc.Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al. (1984) Nature312:604-608; Takeda, S. et al. (1985) Nature 314:452-454).Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to produceAPOP-specific single chain antibodies. Antibodies with relatedspecificity, but of distinct idiotypic composition, may be generated bychain shuffling from random combinatorial immunoglobulin libraries(Burton D. R. (1991) Proc. Natl. Acad. Sci. 88:11120-3).

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening immunoglobulin libraries or panelsof highly specific binding reagents as disclosed in the literature(Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86: 3833-3837; Winter,G. et al. (1991) Nature 349:293-299).

Antibody fragments which contain specific binding sites for APOP mayalso be generated. For example, such fragments include, but are notlimited to, the F(ab')2 fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab)2 fragments.Alternatively, Fab expression libraries may be constructed to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse, W. D. et al. (1989) Science 254:1275-1281).

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding or immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between APOP and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering APOP epitopes is preferred, but a competitivebinding assay may also be employed (Maddox, supra).

In another embodiment of the invention, the polynucleotides encodingAPOP, or any fragment or complement thereof, may be used for therapeuticpurposes. In one aspect, the complement of the polynucleotide encodingAPOP may be used in situations in which it would be desirable to blockthe transcription of the mRNA. In particular, cells may be transformedwith sequences complementary to polynucleotides encoding APOP. Thus,complementary molecules or fragments may be used to modulate APOPactivity, or to achieve regulation of gene function. Such technology isnow well known in the art, and sense or antisense oligonucleotides orlarger fragments, can be designed from various locations along thecoding or control regions of sequences encoding APOP.

Expression vectors derived from retro viruses, adenovirus, herpes orvaccinia viruses, or from various bacterial plasmids may be used fordelivery of nucleotide sequences to the targeted organ, tissue or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct vectors which will express nucleic acid sequencewhich is complementary to the polynucleotides of the gene encoding APOP.These techniques are described both in Sambrook et al. (supra) and inAusubel et al. (supra).

Genes encoding APOP can be turned off by transforming a cell or tissuewith expression vectors which express high levels of a polynucleotide orfragment thereof which encodes APOP. Such constructs may be used tointroduce untranslatable sense or antisense sequences into a cell. Evenin the absence of integration into the DNA, such vectors may continue totranscribe RNA molecules until they are disabled by endogenousnucleases. Transient expression may last for a month or more with anon-replicating vector and even longer if appropriate replicationelements are part of the vector system.

As mentioned above, modifications of gene expression can be obtained bydesigning complementary sequences or antisense molecules (DNA, RNA, orPNA) to the control, 5' or regulatory regions of the gene encoding APOP(signal sequence, promoters, enhancers, and introns). Oligonucleotidesderived from the transcription initiation site, e.g., between positions-10 and +10 from the start site, are preferred. Similarly, inhibitioncan be achieved using "triple helix" base-pairing methodology. Triplehelix pairing is useful because it causes inhibition of the ability ofthe double helix to open sufficiently for the binding of polymerases,transcription factors, or regulatory molecules. Recent therapeuticadvances using triplex DNA have been described in the literature (Gee,J. E. et al. (1994) In: Huber, B. E. and B. I. Carr, Molecular andImmunologic Approaches, Futura Publishing Co., Mt. Kisco, N.Y.). Thecomplementary sequence or antisense molecule may also be designed toblock translation of mRNA by preventing the transcript from binding toribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to catalyze thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Exampleswhich may be used include engineered hammerhead motif ribozyme moleculesthat can specifically and efficiently catalyze endonucleolytic cleavageof sequences encoding APOP.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

Complementary ribonucleic acid molecules and ribozymes of the inventionmay be prepared by any method known in the art for the synthesis ofnucleic acid molecules. These include techniques for chemicallysynthesizing oligonucleotides such as solid phase phosphoramiditechemical synthesis. Alternatively, RNA molecules may be generated by invitro and in vivo transcription of DNA sequences encoding APOP. Such DNAsequences may be incorporated into a wide variety of vectors withsuitable RNA polymerase promoters such as T7 or SP6. Alternatively,these cDNA constructs that synthesize complementary RNA constitutivelyor inducibly can be introduced into cell lines, cells, or tissues.

RNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5' and/or 3' ends of the moleculeor the use of phosphorothioate or 2' O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

Many methods for introducing vectors into cells or tissues are availableand equally suitable for use in vivo, in vitro, and ex vivo. For ex vivotherapy, vectors may be introduced into stem cells taken from thepatient and clonally propagated for autologous transplant back into thatsame patient. Delivery by transfection, by liposome injections orpolycationic amino polymers (Goldman, C. K. et al. (1997) NatureBiotechnology 15:462-66; incorporated herein by reference) may beachieved using methods which are well known in the art.

Any of the therapeutic methods described above may be applied to anysubject in need of such therapy, including, for example, mammals such asdogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

An additional embodiment of the invention relates to the administrationof a pharmaceutical composition, in conjunction with a pharmaceuticallyacceptable carrier, for any of the therapeutic effects discussed above.Such pharmaceutical compositions may consist of APOP, antibodies toAPOP, mimetics, agonists, antagonists, or inhibitors of APOP. Thecompositions may be administered alone or in combination with at leastone other agent, such as stabilizing compound, which may be administeredin any sterile, biocompatible pharmaceutical carrier, including, but notlimited to, saline, buffered saline, dextrose, and water. Thecompositions may be administered to a patient alone, or in combinationwith other agents, drugs or hormones.

The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically-acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Furtherdetails on techniques for formulation and administration may be found inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing Co., Easton, Pa.).

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillers,such as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose, suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

Dragee cores may be used in conjunction with suitable coatings, such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating, such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with a filler or binders, such aslactose or starches, lubricants, such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active compounds maybe dissolved or suspended in suitable liquids, such as fatty oils,liquid, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration maybe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks' solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions maycontain substances which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils such as sesame oil, or synthetic fatty acid esters, such asethyl oleate or triglycerides, or liposomes. Non-lipid polycationicamino polymers may also be used for delivery. Optionally, the suspensionmay also contain suitable stabilizers or agents which increase thesolubility of the compounds to allow for the preparation of highlyconcentrated solutions.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at apH range of 4.5 to 5.5, that is combined with buffer prior to use.

After pharmaceutical compositions have been prepared, they can be placedin an appropriate container and labeled for treatment of an indicatedcondition. For administration of APOP, such labeling would includeamount, frequency, and method of administration.

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells, orin animal models, usually mice, rabbits, dogs, or pigs. The animal modelmay also be used to determine the appropriate concentration range androute of administration. Such information can then be used to determineuseful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of activeingredient, for example APOP or fragments thereof, antibodies of APOP,agonists, antagonists or inhibitors of APOP, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED50 (the dose therapeutically effective in50% of the population) and LD50 (the dose lethal to 50% of thepopulation). The dose ratio between therapeutic and toxic effects is thetherapeutic index, and it can be expressed as the ratio, LD50/ED50.Pharmaceutical compositions which exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesis used in formulating a range of dosage for human use. The dosagecontained in such compositions is preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to atotal dose of about 1 g, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature and generally available to practitioners in the art.Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc.

DIAGNOSTICS

In another embodiment, antibodies which specifically bind APOP may beused for the diagnosis of conditions or diseases characterized byexpression of APOP, or in assays to monitor patients being treated withAPOP, agonists, antagonists or inhibitors. The antibodies useful fordiagnostic purposes may be prepared in the same manner as thosedescribed above for therapeutics. Diagnostic assays for APOP includemethods which utilize the antibody and a label to detect APOP in humanbody fluids or extracts of cells or tissues. The antibodies may be usedwith or without modification, and may be labeled by joining them, eithercovalently or non-covalently, with a reporter molecule. A wide varietyof reporter molecules which are known in the art may be used, several ofwhich are described above.

A variety of protocols including ELISA, RIA, and FACS for measuring APOPare known in the art and provide a basis for diagnosing altered orabnormal levels of APOP expression. Normal or standard values for APOPexpression are established by combining body fluids or cell extractstaken from normal mammalian subjects, preferably human, with antibody toAPOP under conditions suitable for complex formation. The amount ofstandard complex formation may be quantified by various methods, butpreferably by photometric means. Quantities of APOP expressed in subjectsamples, control and disease, from biopsied tissues are compared withthe standard values. Deviation between standard and subject valuesestablishes the parameters for diagnosing disease.

In another embodiment of the invention, the polynucleotides encodingAPOP may be used for diagnostic purposes. The polynucleotides which maybe used include oligonucleotide sequences, complementary RNA and DNAmolecules, and PNAs. The polynucleotides may be used to detect andquantitate gene expression in biopsied tissues in which expression ofAPOP may be correlated with disease. The diagnostic assay may be used todistinguish between absence, presence, and excess expression of APOP,and to monitor regulation of APOP levels during therapeuticintervention.

In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding APOP or closely related molecules, may be used to identifynucleic acid sequences which encode APOP. The specificity of the probe,whether it is made from a highly specific region, e.g., 10 uniquenucleotides in the 5' regulatory region, or a less specific region,e.g., especially in the 3' coding region, and the stringency of thehybridization or amplification (maximal, high, intermediate, or low)will determine whether the probe identifies only naturally occurringsequences encoding APOP, alleles, or related sequences.

Probes may also be used for the detection of related sequences, andshould preferably contain at least 50% of the nucleotides from any ofthe APOP encoding sequences. The hybridization probes of the subjectinvention may be DNA or RNA and derived from the nucleotide sequence ofSEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or from genomic sequenceincluding promoter, enhancer elements, and introns of the naturallyoccurring APOP.

Means for producing specific hybridization probes for DNAs encoding APOPinclude the cloning of nucleic acid sequences encoding APOP or APOPderivatives into vectors for the production of mRNA probes. Such vectorsare known in the art, commercially available, and may be used tosynthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, radionuclides such as 32P or 35S, or enzymatic labels, such asalkaline phosphatase coupled to the probe via avidin/biotin couplingsystems, and the like.

Polynucleotide sequences encoding APOP may be used for the diagnosis ofconditions or disorders which are associated with expression of APOP.Examples of such conditions or disorders include, but are not limitedto, cancers such as adenocarcinoma, leukemia, lymphoma, melanoma,myeloma, sarcoma, and teratocarcinoma, and particularly, cancers of theadrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gallbladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung,muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands,skin, spleen, testis, thymus, thyroid, and uterus; disorders withassociated inflammation such as Addison's disease, adult respiratorydistress syndrome, allergies, anemia, asthma, atherosclerosis,bronchitis, cholecystitis, Crohn's disease, ulcerative colitis, atopicdermatitis, dermatomyositis, diabetes mellitus, emphysema, atrophicgastritis, glomerulonephritis, gout, Graves' disease, hypereosinophilia,irritable bowel syndrome, lupus erythematosus, multiple sclerosis,myasthenia gravis, myocardial or pericardialinflammation,osteoarthritis, osteoporosis, pancreatitis, polymyositis,rheumatoid arthritis, scleroderma, Sjogren's syndrome, and autoimmunethyroiditis; complications of cancer, hemodialysis, extracorporealcirculation; viral, bacterial, fungal, parasitic, protozoal, andhelminthic infections and trauma; disorders with associated apoptosissuch as AIDS and other infectious or genetic immunodeficiencies,neurodegenerative diseases such as Alzheimer's disease, Parkinson'sdisease, amyotrophic lateral sclerosis, retinitis pigmentosa, andcerebellar degeneration, myelodysplastic syndromes such as aplasticanemia, ischemic injuries such as myocardial infarction, stroke, andreperfusion injury, toxin-induced diseases such as alcohol-induced liverdamage, cirrhosis, and lathyrism, wasting diseases such as cachexia,viral infections such as those caused by hepatitis B and C, andosteoporosis. The polynucleotide sequences encoding APOP may be used inSouthern or northern analysis, dot blot, or other membrane-basedtechnologies; in PCR technologies; or in dipstick, pin, ELISA assays ormicroarrays utilizing fluids or tissues from patient biopsies to detectaltered APOP expression. Such qualitative or quantitative methods arewell known in the art.

In a particular aspect, the nucleotide sequences encoding APOP may beuseful in assays that detect activation or induction of various cancers,particularly those mentioned above. The nucleotide sequences encodingAPOP may be labeled by standard methods, and added to a fluid or tissuesample from a patient under conditions suitable for the formation ofhybridization complexes. After a suitable incubation period, the sampleis washed and the signal is quantitated and compared with a standardvalue. If the amount of signal in the biopsied or extracted sample issignificantly altered from that of a comparable control sample, thenucleotide sequences have hybridized with nucleotide sequences in thesample, and the presence of altered levels of nucleotide sequencesencoding APOP in the sample indicates the presence of the associateddisease. Such assays may also be used to evaluate the efficacy of aparticular therapeutic treatment regimen in animal studies, in clinicaltrials, or in monitoring the treatment of an individual patient.

In order to provide a basis for the diagnosis of disease associated withexpression of APOP, a normal or standard profile for expression isestablished. This may be accomplished by combining body fluids or cellextracts taken from normal subjects, either animal or human, with asequence, or a fragment thereof, which encodes APOP, under conditionssuitable for hybridization or amplification. Standard hybridization maybe quantified by comparing the values obtained from normal subjects withthose from an experiment where a known amount of a substantiallypurified polynucleotide is used. Standard values obtained from normalsamples may be compared with values obtained from samples from patientswho are symptomatic for disease. Deviation between standard and subjectvalues is used to establish the presence of disease.

Once disease is established and a treatment protocol is initiated,hybridization assays may be repeated on a regular basis to evaluatewhether the level of expression in the patient begins to approximatethat which is observed in the normal patient. The results obtained fromsuccessive assays may be used to show the efficacy of treatment over aperiod ranging from several days to months.

With respect to cancer, the presence of a relatively high amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Additional diagnostic uses for oligonucleotides designed from thesequences encoding APOP may involve the use of PCR. Such oligomers maybe chemically synthesized, generated enzymatically, or produced invitro. Oligomers will preferably consist of two nucleotide sequences,one with sense orientation (5'→3') and another with antisense (3'←5'),employed under optimized conditions for identification of a specificgene or condition. The same two oligomers, nested sets of oligomers, oreven a degenerate pool of oligomers may be employed under less stringentconditions for detection and/or quantitation of closely related DNA orRNA sequences.

Methods which may also be used to quantitate the expression of APOPinclude radiolabeling or biotinylating nucleotides, coamplification of acontrol nucleic acid, and standard curves onto which the experimentalresults are interpolated (Melby, P. C. et al. 1993) J. Immunol. Meth.,159:235-244; Duplaa, C. et al. (1993) Anal. Biochem. 211:229-236). Thespeed of quantitation of multiple samples may be accelerated by runningthe assay in an ELISA format where the oligomer of interest is presentedin various dilutions and a spectrophotometric or calorimetric responsegives rapid quantitation.

In further embodiments, an oligonucleotide derived from any of thepolynucleotide sequences described herein may be used as a target in amicroarray. The microarray can be used to monitor the expression levelof large numbers of genes simultaneously (to produce a transcriptimage), and to identify genetic variants, mutations, and polymorphisms.This information will be useful in determining gene function,understanding the genetic basis of disease, diagnosing disease, and indeveloping and monitoring the activity of therapeutic agents

In one embodiment, the microarray is prepared and used according to themethods known in the art such as those described in PCT applicationWO95/11995 (Chee et al.), Lockhart, D. J. et al. (1996; Nat. Biotech.14: 1675-1680) and Schena, M. et al. (1996; Proc. Natl. Acad. Sci.93:10614-10619).

The microarray is preferably composed of a large number of unique,single-stranded nucleic acid sequences, usually either syntheticantisense oligonucleotides or fragments of cDNAs, fixed to a solidsupport. The oligonucleotides are preferably about 6-60 nucleotides inlength, more preferably about 15 to 30 nucleotides in length, and mostpreferably about 20 to 25 nucleotides in length. For a certain type ofmicroarray, it may be preferable to use oligonucleotides which are only7 to 10 nucleotides in length. The microarray may containoligonucleotides which cover the known 5' (or 3') sequence, or maycontain sequential oligonucleotides which cover the full lengthsequence; or unique oligonucleotides selected from particular areasalong the length of the sequence. Polynucleotides used in the microarraymay be oligonucleotides that are specific to a gene or genes of interestin which at least a fragment of the sequence is known or that arespecific to one or more unidentified cDNAs which are common to aparticular cell or tissue type or to a normal, developmental, or diseasestate. In certain situations, it may be appropriate to use pairs ofoligonucleotides on a microarray. The pairs will be identical, exceptfor one nucleotide preferably located in the center of the sequence. Thesecond oligonucleotide in the pair (mismatched by one) serves as acontrol. The number of oligonucleotide pairs may range from 2 to1,000,000.

In order to produce oligonucleotides to a known sequence for amicroarray, the gene of interest is examined using a computer algorithmwhich starts at the 5' or more preferably at the 3' end of thenucleotide sequence. The algorithm identifies oligomers of definedlength that are unique to the gene, have a GC content within a rangesuitable for hybridization, and lack predicted secondary structure thatmay interfere with hybridization. In one aspect, the oligomers aresynthesized at designated areas on a substrate using a light-directedchemical process. The substrate may be paper, nylon or any other type ofmembrane, filter, chip, glass slide, or any other suitable solidsupport.

In one aspect, the oligonucleotides may be synthesized on the surface ofthe substrate by using a chemical coupling procedure and an ink jetapplication apparatus, such as that described in PCT applicationWO95/251 116 (Baldeschweiler et al.). In another aspect, a "gridded"array analogous to a dot or slot blot (HYBRIDOT® apparatus, GIBCO/BRL)may be used to arrange and link cDNA fragments or oligonucleotides tothe surface of a substrate using a vacuum system, thermal, UV,mechanical or chemical bonding procedures. In yet another aspect, anarray may be produced by hand or by using available devices, materials,and machines (including Brinkmann® multichannel pipettors or roboticinstruments) and may contain 8, 24, 96, 384, 1536 or 6144oligonucleotides, or any other multiple from 2 to 1,000,000 which lendsitself to the efficient use of commercially available instrumentation.

In order to conduct sample analysis using the microarrays,polynucleotides are extracted from a biological sample. The biologicalsamples may be obtained from any bodily fluid (blood, urine, saliva,phlegm, gastric juices, etc.), cultured cells, biopsies, or other tissuepreparations. To produce probes, the polynucleotides extracted from thesample are used to produce nucleic acid sequences which arecomplementary to the nucleic acids on the microarray. If the microarrayconsists of cDNAs, antisense RNAs (aRNA) are appropriate probes.Therefore, in one aspect, mRNA is used to produce cDNA which, in turnand in the presence of fluorescent nucleotides, is used to producefragment or oligonucleotide aRNA probes. These fluorescently labeledprobes are incubated with the microarray so that the probe sequenceshybridize to the cDNA oligonucleotides of the microarray. In anotheraspect, nucleic acid sequences used as probes can includepolynucleotides, fragments, and complementary or antisense sequencesproduced using restriction enzymes, PCR technologies, and Oligolabelingor TransProbe kits (Pharmacia) well known in the area of hybridizationtechnology.

Incubation conditions are adjusted so that hybridization occurs withprecise complementary matches or with various degrees of lesscomplementarity. After removal of nonhybridized probes, a scanner isused to determine the levels and patterns of fluorescence. The scannedimages are examined to determine degree of complementarity and therelative abundance of each oligonucleotide sequence on the microarray. Adetection system may be used to measure the absence, presence, andamount of hybridization for all of the distinct sequencessimultaneously. This data may be used for large scale correlationstudies or functional analysis of the sequences, mutations, variants, orpolymorphisms among samples (Heller, R. A. et al., (1997) Proc. Natl.Acad. Sci. 94:2150-55).

In another embodiment of the invention, the nucleic acid sequences whichencode APOP may also be used to generate hybridization probes which areuseful for mapping the naturally occurring genomic sequence. Thesequences may be mapped to a particular chromosome, to a specific regionof a chromosome or to artificial chromosome constructions, such as humanartificial chromosomes (HACs), yeast artificial chromosomes (YACs),bacterial artificial chromosomes (BACs), bacterial P1 constructions orsingle chromosome cDNA libraries as reviewed in Price, C. M. (1993)Blood Rev. 7:127-134, and Trask, B. J. (1991) Trends Genet. 7:149-154.

Fluorescent in situ hybridization (FISH as described in Verma et al.(1988) Human Chromosomes: A Manual of Basic Techniques, Pergamon Press,New York, N.Y.) may be correlated with other physical chromosome mappingtechniques and genetic map data. Examples of genetic map data can befound in various scientific journals or at Online Mendelian Inheritancein Man (OMIM). Correlation between the location of the gene encodingAPOP on a physical chromosomal map and a specific disease , orpredisposition to a specific disease, may help delimit the region of DNAassociated with that genetic disease. The nucleotide sequences of thesubject invention may be used to detect differences in gene sequencesbetween normal, carrier, or affected individuals.

In situ hybridization of chromosomal preparations and physical mappingtechniques such as linkage analysis using established chromosomalmarkers may be used for extending genetic maps. Often the placement of agene on the chromosome of another mammalian species, such as mouse, mayreveal associated markers even if the number or arm of a particularhuman chromosome is not known. New sequences can be assigned tochromosomal arms, or parts thereof, by physical mapping. This providesvaluable information to investigators searching for disease genes usingpositional cloning or other gene discovery techniques. Once the diseaseor syndrome has been crudely localized by genetic linkage to aparticular genomic region, for example, AT to 11q22-23 (Gatti, R. A. etal. (1988) Nature 336:577-580), any sequences mapping to that area mayrepresent associated or regulatory genes for further investigation. Thenucleotide sequence of the subject invention may also be used to detectdifferences in the chromosomal location due to translocation, inversion,etc. among normal, carrier, or affected individuals.

In another embodiment of the invention, APOP, its catalytic orimmunogenic fragments or oligopeptides thereof, can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes, betweenAPOP and the agent being tested, may be measured.

Another technique for drug screening which may be used provides for highthroughput screening of compounds having suitable binding affinity tothe protein of interest as described in published PCT applicationWO84/03564. In this method, as applied to APOP large numbers ofdifferent small test compounds are synthesized on a solid substrate,such as plastic pins or some other surface. The test compounds arereacted with APOP, or fragments thereof, and washed. Bound APOP is thendetected by methods well known in the art. Purified APOP can also becoated directly onto plates for use in the aforementioned drug screeningtechniques. Alternatively, non-neutralizing antibodies can be used tocapture the peptide and immobilize it on a solid support.

In another embodiment, one may use competitive drug screening assays inwhich neutralizing antibodies capable of binding APOP specificallycompete with a test compound for binding APOP. In this manner, theantibodies can be used to detect the presence of any peptide whichshares one or more antigenic determinants with APOP.

In additional embodiments, the nucleotide sequences which encode APOPmay be used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

The examples below are provided to illustrate the subject invention andare not included for the purpose of limiting the invention.

EXAMPLES

I cDNA Library Construction

The SYNORAB01 cDNA library was constructed from RNA isolated from hipsynovial tissue removed from a 68 year old Caucasian with rheumatoidarthritis during hip replacement surgery. The frozen tissue was groundin a mortar and pestle and lysed immediately in a buffer containingguanidinium isothiocyanate. The lysate was extracted with acid phenoland centrifuged over a 5.7 M CsCl cushion using a Beckman SW28 rotor ina Beckman L8-70M Ultracentrifuge (Beckman Instruments) for 18 hours at25,000 rpm at ambient temperature. The RNA was precipitated using 0.3 Msodium acetate and 2.5 volumes of ethanol, resuspended in water andDNase treated for 15 min at 37° C. The RNA was isolated using the QiagenOligotex kit (QIAGEN, Chatsworth, Calif.) and used to construct the cDNAlibrary.

cDNA synthesis was primed using a combination of oligo d(T) and randomprimers, and synthetic adaptor oligonucleotides were ligated onto thecDNA ends to enable insertion into the Uni-ZAP™ vector system(Stratagene). E. coli host strain XL1-Blue® (Stratagene) wasco-transfected with phagemid and f1 helper phage particles. Proteinsderived from both the lambda phage and f1 helper phage initiated new DNAsynthesis from defined sequences on the lambda target DNA to create thesmaller, single-stranded circular pBluescript® phagemid (Stratagene)which contains the SYNORAB01 inserts. When the phagemid DNA was releasedfrom the cells, it was purified and used to reinfect fresh bacterialhost cells (SOLR™; Stratagene). Transformed bacteria expressing theβ-lactamase gene on the phagemid survived selection on medium containingampicillin and produced double-stranded phagemid.

The LATRTUT02 cDNA library was constructed from cancerous heart tissueremoved from a 43-year-old Caucasian male who had undergone annuloplastyfollowing diagnosis of atrial myxoma. The frozen tissue was homogenizedand lysed using a Brinkmann Homogenizer Polytron PT-3000 (BrinkmannInstruments, Westbury, N.J.) in guanidinium isothiocyanate solution. Thelysate was centrifuged over a 5.7 M CsCl cushion using an Beckman SW28rotor in a Beckman L8-70M Ultracentrifuge (Beckman Instruments) for 18hours at 25,000 rpm at ambient temperature. The RNA was extracted withacid phenol pH 4.7, precipitated using 0.3 M sodium acetate and 2.5volumes of ethanol, resuspended in RNAse-free water, and DNase treatedat 37° C. The RNA extraction was repeated with acid phenol pH 4.7 andprecipitated with sodium acetate and ethanol as before. The mRNA wasthen isolated using the Qiagen Oligotex kit (QIAGEN) and used toconstruct the cDNA library.

The OVARTUT01 cDNA library was constructed from tumorous ovary tissueobtained by salpingo-oophorectomy of a 43 year old Caucasian female toremove an ovary which had been diagnosed with a malignant neoplasm. Thefrozen tissue was homogenized and lysed using a Brinkmann HomogenizerPolytron PT-3000 (Brinkmann Instruments) in guanidinium isothiocyanatesolution. The lysate was centrifuged over a 5.7 M CsCl cushion using anBeckman SW28 rotor in a Beckman L8-70M Ultracentrifuge (BeckmanInstruments) for 18 hours at 25,000 rpm at ambient temperature. The RNAwas extracted with acid phenol pH 4.0, precipitated using 0.3 M sodiumacetate and 2.5 volumes of ethanol, resuspended in RNAse-free water andDNase treated at 37° C. The RNA extraction and precipitation wererepeated as before. The mRNA was then isolated using the Qiagen Oligotexkit (QIAGEN) and used to construct the cDNA library.

The mRNA was handled according to the recommended protocols in theSuperScript Plasmid System for cDNA Synthesis and Plasmid Cloning (Cat.#18248-013,GIBCO/BRL). The cDNAs were fractionated on a Sepharose CL4Bcolumn (Cat. #275105-01; Pharmacia), and those cDNAs exceeding 400 bpwere ligated into pINCY 1 (LATRTUT02) or pSport I (OVARTUT01). Theplasmids were subsequently transformed into DH5a™ competent cells (Cat.#18258-012; GIBCO/BRL).

II Isolation and Sequencing of cDNA Clones

Phagemid DNA for SYNORAB01 was purified using the QIAWELL-8 plasmidpurification system (QIAGEN) and prepared for sequencing. Chaintermination reaction products were electrophoresed onurea-polyacrylamide gels and detected by fluorescence.

Plasmid cDNA for LATRTUT02 or OVARTUT01 was released from the cells andpurified using the REAL Prep 96 Plasmid Kit (Catalog #26173, QIAGEN).This kit enabled the simultaneous purification of 96 samples in a96-well block using multi-channel reagent dispensers. The recommendedprotocol was employed except for the following changes: 1) the bacteriawere cultured in 1 ml of sterile Terrific Broth (Catalog #22711,GIBCO/BRL) with carbenicillin at 25 mg/L and glycerol at 0.4%; 2) afterinoculation, the cultures were incubated for 19 hours and at the end ofincubation, the cells were lysed with 0.3 ml of lysis buffer; and 3)following isopropanol precipitation, the plasmid DNA pellet wasresuspended in 0.1 ml of distilled water. After the last step in theprotocol, samples were transferred to a 96-well block for storage at 4°C.

The cDNAs for all three libraries were sequenced according to the methodof Sanger et al. (1975, J. Mol. Biol. 94:441f), using the Perkin ElmerCatalyst 800 or a Hamilton Micro Lab 2200 (Hamilton, Reno, Nev.) incombination with Peltier Thermal Cyclers (PTC200 from MJ Research,Watertown, Mass.) and Applied Biosystems 377 DNA Sequencing Systems orthe Perkin Elmer 373 DNA Sequencing System and the reading frame wasdetermined.

III Homology Searching of cDNA Clones and Their Deduced Proteins

The nucleotide sequences of the Sequence Listing or amino acid sequencesdeduced from them were used as query sequences against databases such asGenBank, SwissProt, BLOCKS, and Pima II. These databases which containpreviously identified and annotated sequences were searched for regionsof homology (similarity) using BLAST, which stands for Basic LocalAlignment Search Tool (Altschul SF (1993) J. Mol. Evol. 36:290-300;Altschul, SF et al. (1990) J. Mol. Biol. 215:403-10).

BLAST produces alignments of both nucleotide and amino acid sequences todetermine sequence similarity. Because of the local nature of thealignments, BLAST is especially useful in determining exact matches orin identifying homologs which may be of prokaryotic (bacterial) oreukaryotic (animal, fungal or plant) origin. Other algorithms such asthe one described in Smith RF and TF Smith (1992 Protein Engineering5:35-51), incorporated herein by reference, can be used when dealingwith primary sequence patterns and secondary structure gap penalties. Asdisclosed in this application, the sequences have lengths of at least 49nucleotides, and no more than 12% uncalled bases (where N is recordedrather than A, C, G, or T).

The BLAST approach, as detailed in Karlin and Altschul (1993; Proc NatAcad Sci 90:5873-7) and incorporated herein by reference, searchesmatches between a query sequence and a database sequence, to evaluatethe statistical significance of any matches found, and to report onlythose matches which satisfy the user-selected threshold of significance.In this application, threshold was set at 10⁻²⁵ for nucleotides and10⁻¹⁴ for peptides.

IV Northern Analysis

Northern analysis is a laboratory technique used to detect the presenceof a transcript of a gene and involves the hybridization of a labelednucleotide sequence to a membrane on which RNAs from a particular celltype or tissue have been bound (Sambrook et al., supra).

Analogous computer techniques using BLAST (Altschul, S. F. (1993)J.Mol.Evol. 36:290-300; Altschul, S. F. et al. (1990) J.Mol.Evol.215:403-410) are used to search for identical or related molecules innucleotide databases such as GenBank or the LIFESEQ™ database (IncytePharmaceuticals). This analysis is much faster than multiple,membrane-based hybridizations. In addition, the sensitivity of thecomputer search can be modified to determine whether any particularmatch is categorized as exact or homologous.

The basis of the search is the product score which is defined as:

    % sequence identity×% maximum BLAST score/100

The product score takes into account both the degree of similaritybetween two sequences and the length of the sequence match. For example,with a product score of 40, the match will be exact within a 1-2% error;and at 70, the match will be exact. Homologous molecules are usuallyidentified by selecting those which show product scores between 15 and40, although lower scores may identify related molecules.

The results of northern analysis are reported as a list of libraries inwhich the transcript encoding APOP occurs. Abundance and percentabundance are also reported. Abundance directly reflects the number oftimes a particular transcript is represented in a cDNA library, andpercent abundance is abundance divided by the total number of sequencesexamined in the cDNA library.

V Extension of APOP Encoding Polynucleotides

The nucleic acid sequence of the Incyte Clones 358673, 1352286, or815087 was used to design oligonucleotide primers for extending apartial nucleotide sequence to full length. One primer was synthesizedto initiate extension in the antisense direction, and the other wassynthesized to extend sequence in the sense direction. Primers were usedto facilitate the extension of the known sequence "outward" generatingamplicons containing new, unknown nucleotide sequence for the region ofinterest. The initial primers were designed from the cDNA using OLIGO4.06 (National Biosciences), or another appropriate program, to be about22 to about 30 nucleotides in length, to have a GC content of 50% ormore, and to anneal to the target sequence at temperatures of about 68°to about 72° C. Any stretch of nucleotides which would result in hairpinstructures and primer-primer dimerizations was avoided.

Selected human cDNA libraries (GIBCO/BRL) were used to extend thesequence. If more than one extension is necessary or desired, additionalsets of primers are designed to further extend the known region.

High fidelity amplification was obtained by following the instructionsfor the XL-PCR kit (Perkin Elmer) and thoroughly mixing the enzyme andreaction mix. Beginning with 40 pmol of each primer and the recommendedconcentrations of all other components of the kit, PCR was performedusing the Peltier Thermal Cycler (PTC200; M.J. Research, Watertown,Mass.) and the following parameters:

    ______________________________________                                        Step 1       94° C. for 1 min (initial denaturation)                     Step 2 65° C. for 1 min                                                Step 3 68° C. for 6 min                                                Step 4 94° C. for 15 sec                                               Step 5 65° C. for 1 min                                                Step 6 68° C. for 7 min                                                Step 7 Repeat step 4-6 for 15 additional cycles                               Step 8 94° C. for 15 sec                                               Step 9 65° C. for 1 min                                                Step 10 68° C. for 7:15 min                                            Step 11 Repeat step 8-10 for 12 cycles                                        Step 12 72° C. for 8 min                                               Step 13 4° C. (and holding)                                          ______________________________________                                    

A 5-10 μl aliquot of the reaction mixture was analyzed byelectrophoresis on a low concentration (about 0.6-0.8%) agarose mini-gelto determine which reactions were successful in extending the sequence.Bands thought to contain the largest products were excised from the gel,purified using QIAQuick™ (QIAGEN), and trimmed of overhangs using Klenowenzyme to facilitate religation and cloning.

After ethanol precipitation, the products were redissolved in 13 μl ofligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase were added, and the mixture was incubated at roomtemperature for 2-3 hours or overnight at 16° C. Competent E. coli cells(in 40 μl of appropriate media) were transformed with 3 μl of ligationmixture and cultured in 80 μl of SOC medium (Sambrook et al., supra).After incubation for one hour at 37° C., the E. coli mixture was platedon Luria Bertani (LB)-agar (Sambrook et al., supra) containing 2× Carb.The following day, several colonies were randomly picked from each plateand cultured in 150 μl of liquid LB/2× Carb medium placed in anindividual well of an appropriate, commercially-available, sterile96-well microtiter plate. The following day, 5 μl of each overnightculture was transferred into a non-sterile 96-well plate and afterdilution 1:10 with water, 5 μl of each sample was transferred into a PCRarray.

For PCR amplification, 18 μl of concentrated PCR reaction mix (3.3×)containing 4 units of rTth DNA polymerase, a vector primer, and one orboth of the gene specific primers used for the extension reaction wereadded to each well. Amplification was performed using the followingconditions:

    ______________________________________                                        Step 1     94° C. for 60 sec                                             Step 2 94° C. for 20 sec                                               Step 3 55° C. for 30 sec                                               Step 4 72° C. for 90 sec                                               Step 5 Repeat steps 2-4 for an additional 29 cycles                           Step 6 72° C. for 180 sec                                              Step 7  4° C. (and holding)                                          ______________________________________                                    

Aliquots of the PCR reactions were run on agarose gels together withmolecular weight markers. The sizes of the PCR products were compared tothe original partial cDNAs, and appropriate clones were selected,ligated into plasmid, and sequenced.

In like manner, the nucleotide sequence of SEQ ID NO:2, SEQ ID NO:4, orSEQ ID NO:6 is used to obtain 5' regulatory sequences using theprocedure above, oligonucleotides designed for 5' extension, and anappropriate genomic library.

VI Labeling and Use of Individual Hybridization Probes

Hybridization probes derived from SEQ ID NO:2, SEQ ID NO:4, or SEQ IDNO:6 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although thelabeling of oligonucleotides, consisting of about 20 base-pairs, isspecifically described, essentially the same procedure is used withlarger nucleotide fragments. Oligonucleotides are designed usingstate-of-the-art software such as OLIGO 4.06 (National Biosciences),labeled by combining 50 pmol of each oligomer and 250 μCi of [γ-³² P]adenosine triphosphate (Amersham) and T4 polynucleotide kinase (DuPontNEN®, Boston, Mass.). The labeled oligonucleotides are substantiallypurified with Sephadex G-25 superfine resin column (Pharmacia & Upjohn).A aliquot containing 10⁷ counts per minute of the labeled probe is usedin a typical membrane-based hybridization analysis of human genomic DNAdigested with one of the following endonucleases (Ase I, Bgl II, Eco RI,Pst I, Xba 1, or Pvu II; DuPont NEN®).

The DNA from each digest is fractionated on a 0.7 percent agarose geland transferred to nylon membranes (Nytran Plus, Schleicher & Schuell,Durham, N.H.). Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1× salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR™ film(Kodak, Rochester, N.Y.) is exposed to the blots in a Phosphoimagercassette (Molecular Dynamics, Sunnyvale, Calif.) for several hours,hybridization patterns are compared visually.

VII Microarrays

To produce oligonucleotides for a microarray, one of the nucleotidesequences of the present invention are examined using a computeralgorithm which starts at the 3' end of the nucleotide sequence. Thealgorithm identified oligomers of defined length that are unique to thegene, have a GC content within a range suitable for hybridization, andlack predicted secondary structure that would interfere withhybridization. The algorithm identifies approximately 20sequence-specific oligonucleotides of 20 nucleotides in length(20-mers). A matched set of oligonucleotides are created in which onenucleotide in the center of each sequence is altered. This processisrepeated for each gene in the microarray, and double sets of twenty 20mers are synthesized and arranged on the surface of the silicon chipusing a light-directed chemical process, such as that discussed in Chee,supra.

In the alternative, a chemical coupling procedure and an ink jet deviceare used to synthesize oligomers on the surface of a substrate (cf.Baldeschweiler, supra). In another alternative, a "gridded" arrayanalogous to a dot (or slot) blot is used to arrange and link cDNAfragments or oligonucleotides to the surface of a substrate using avacuum system, thermal, UV, mechanical or chemical bonding procedures. Atypical array may be produced by hand or using available materials andmachines and contain grids of 8 dots, 24 dots, 96 dots, 384 dots, 1536dots or 6144 dots. After hybridization, the microarray is washed toremove nonhybridized probes, and a scanner is used to determine thelevels and patterns of fluorescence. The scanned image is examined todetermine degree of complementarity and the relativeabundance/expression level of each oligonucleotide sequence in themicroarray.

VIII Complementary Polynucleotides

Sequence complementary to the APOP-encoding sequence, or any partthereof, is used to decrease or inhibit expression of naturallyoccurring APOP. Although use of oligonucleotides comprising from about15 to about 30 base-pairs is described, essentially the same procedureis used with smaller or larger sequence fragments. Appropriateoligonucleotides are designed using Oligo 4.06 software and the codingsequence of APOP, SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5. To inhibittranscription, a complementary oligonucleotide is designed from the mostunique 5' sequence and used to prevent promoter binding to the codingsequence. To inhibit translation, a complementary oligonucleotide isdesigned to prevent ribosomal binding to the APOP-encoding transcript.

IX Expression of APOP

Expression of APOP is accomplished by subcloning the cDNAs intoappropriate vectors and transforming the vectors into host cells. Inthis case, the cloning vector is also used to express APOP in E. coli.Upstream of the cloning site, this vector contains a promoter forβ-galactosidase, followed by sequence containing the amino-terminal Met,and the subsequent seven residues of β-galactosidase. Immediatelyfollowing these eight residues is a bacteriophage promoter useful fortranscription and a linker containing a number of unique restrictionsites.

Induction of an isolated, transformed bacterial strain with IPTG usingstandard methods produces a fusion protein which consists of the firsteight residues of β-galactosidase, about 5 to 15 residues of linker, andthe full length protein. The signal residues direct the secretion ofAPOP into the bacterial growth media which can be used directly in thefollowing assay for activity.

X Demonstration of APOP Activity

APOP can be expressed by transforming a mammalian cell line such asCOS7, HeLa or CHO with an eukaryotic expression vector encoding APOP.Eukaryotic expression vectors are commercially available, and thetechniques to introduce them into cells are well known to those skilledin the art. The cells with and without the APOP expression vector areincubated for 48-72 hours after transformation under conditionsappropriate for the cell line to allow expression of APOP. Phasemicroscopy is subsequently used to compare the mitotic index oftransformed versus control cells. An increase in the mitotic index whereAPOP stimulates cell proliferation indicates APOP activity. Likewise, adecrease in cell numbers where APOP stimulates apoptosis indicates APOPactivity.

XI Production of APOP Specific Antibodies

APOP that is substantially purified using PAGE electrophoresis(Sambrook, supra), or other purification techniques, is used to immunizerabbits and to produce antibodies using standard protocols. The aminoacid sequence deduced from SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6 isanalyzed using DNASTAR software (DNASTAR Inc) to determine regions ofhigh immunogenicity and a corresponding oligopeptide is synthesized andused to raise antibodies by means known to those of skill in the art.Selection of appropriate epitopes, such as those near the C-terminus orin hydrophilic regions, is described by Ausubel et al. (supra), andothers.

Typically, the oligopeptides are 15 residues in length, synthesizedusing an Applied Biosystems Peptide Synthesizer Model 431A usingfmoc-chemistry, and coupled to keyhole limpet hemocyanin (KLH, Sigma,St. Louis, Mo.) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimideester (MBS; Ausubel et al., supra). Rabbits are immunized with theoligopeptide-KLH complex in complete Freund's adjuvant. The resultingantiseraare tested for antipeptide activity, for example, by binding thepeptide to plastic, blocking with 1% BSA, reacting with rabbit antisera,washing, and reacting with radio iodinated, goat anti-rabbit IgG.

XII Purification of Naturally Occurring APOP Using Specific Antibodies

Naturally occurring or recombinant APOP is substantially purified byimmunoaffinity chromatography using antibodies specific for APOP. Animmunoaffinity column is constructed by covalently coupling APOPantibody to an activated chromatographic resin, such as CNBr-activatedSepharose (Pharmacia & Upjohn). After the coupling, the resin is blockedand washed according to the manufacturer's instructions.

Media containing APOP is passed over the immunoaffinity column, and thecolumn is washed under conditions that allow the preferential absorbanceof APOP (e.g., high ionic strength buffers in the presence ofdetergent). The column is eluted under conditions that disruptantibody/APOP binding (eg, a buffer of pH 2-3 or a high concentration ofa chaotrope, such as urea or thiocyanate ion), and APOP is collected.

XIII Identification of Molecules Which Interact with APOP

APOP or biologically active fragments thereof are labeled with ¹²⁵ IBolton-Hunter reagent (Bolton et al. (1973) Biochem. J. 133: 529).Candidate molecules previously arrayed in the wells of a multi-wellplate are incubated with the labeled APOP, washed and any wells withlabeled APOP complex are assayed. Data obtained using differentconcentrations of APOP are used to calculate values for the number,affinity, and association of APOP with the candidate molecules.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology or related fields are intended to be within the scopeof the following claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 9                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 168 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: SYNORAB01                                                        (B) CLONE: 358673                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - Met Phe Gln Ile Pro Glu Phe Glu Pro Ser Gl - #u Gln Glu Asp Ser        Ser                                                                              1               5  - #                10  - #                15              - - Ser Ala Glu Arg Gly Leu Gly Pro Ser Pro Al - #a Gly Asp Gly Pro Ser                  20      - #            25      - #            30                   - - Gly Ser Gly Lys His His Arg Gln Ala Pro Gl - #y Leu Leu Trp Asp Ala              35          - #        40          - #        45                       - - Ser His Gln Gln Glu Gln Pro Thr Ser Ser Se - #r His His Gly Gly Ala          50              - #    55              - #    60                           - - Gly Ala Val Glu Ile Arg Ser Arg His Ser Se - #r Tyr Pro Ala Gly Thr      65                  - #70                  - #75                  - #80        - - Glu Asp Asp Glu Gly Met Gly Glu Glu Pro Se - #r Pro Phe Arg Gly Arg                      85  - #                90  - #                95               - - Ser Arg Ser Ala Pro Pro Asn Leu Trp Ala Al - #a Gln Arg Tyr Gly Arg                  100      - #           105      - #           110                  - - Glu Leu Arg Arg Met Ser Asp Glu Phe Val As - #p Ser Phe Lys Lys Gly              115          - #       120          - #       125                      - - Leu Pro Arg Pro Lys Ser Ala Gly Thr Ala Th - #r Gln Met Arg Gln Ser          130              - #   135              - #   140                          - - Ser Ser Trp Thr Arg Val Phe Gln Ser Trp Tr - #p Asp Arg Asn Leu Gly      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Arg Gly Ser Ser Ala Pro Ser Gln                                                          165                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1105 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: 358673                                                           (B) CLONE: SYNORAB01                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - CGACCGTCCG CGGGAGACTG AGGTCCTGAG CCGACAGCCT CAGCTCCCTG CC -            #AGGCCAGA     60                                                                 - - CCCGGCAGAC AGATGAGGGC CCAGGAGGCC TGGCGGGCCT GGGGGCGCTA CG -            #GTGGGAGA    120                                                                 - - GGAAGCCAGG GGTACCTGCC TCTGCCTTCC AGGGCCACCG TTGGCCCCAG CT -            #GTGCCTTG    180                                                                 - - ACTACGTAAC ATCTTGTCCT CACAGCCCAG AGCATGTTCC AGATCCCAGA GT -            #TTGAGCCG    240                                                                 - - AGTGAGCAGG AAGACTCCAG CTCTGCAGAG AGGGGCCTGG GCCCCAGCCC CG -            #CAGGGGAC    300                                                                 - - GGGCCCTCAG GCTCCGGCAA GCATCATCGC CAGGCCCCAG GCCTCCTGTG GG -            #ACGCCAGT    360                                                                 - - CACCAGCAGG AGCAGCCAAC CAGCAGCAGC CATCATGGAG GCGCTGGGGC TG -            #TGGAGATC    420                                                                 - - CGGAGTCGCC ACAGCTCCTA CCCCGCGGGG ACGGAGGACG ACGAAGGGAT GG -            #GGGAGGAG    480                                                                 - - CCCAGCCCCT TTCGGGGCCG CTCGCGCTCG GCGCCCCCCA ACCTCTGGGC AG -            #CACAGCGC    540                                                                 - - TATGGCCGCG AGCTCCGGAG GATGAGTGAC GAGTTTGTGG ACTCCTTTAA GA -            #AGGGACTT    600                                                                 - - CCTCGCCCGA AGAGCGCGGG CACAGCAACG CAGATGCGGC AAAGCTCCAG CT -            #GGACGCGA    660                                                                 - - GTCTTCCAGT CCTGGTGGGA TCGGAACTTG GGCAGGGGAA GCTCCGCCCC CT -            #CCCAGTGA    720                                                                 - - CCTTCGCTCC ACATCCCGAA ACTCCACCCG TTCCCACTGC CCTGGGCAGC CA -            #TCTTGAAT    780                                                                 - - ATGGGCGGAA GTACTTCCCT CAGGCCTATG CAAAAAGAGG ATCCGTGCTG TC -            #TCCTTTGG    840                                                                 - - AGGGAGGGCT GACCCAGATT CCCTTCCGGT GCGTGTGAAG CCACGGAAGG CT -            #TGGTCCCA    900                                                                 - - TCGGAAGTTT TGGGTTTTCC GCCCACAGCC GCCGGAAGTG GCTCCGTGGC CC -            #CGCCCTCA    960                                                                 - - GGCTCCGGGC TTTCCCCCAG GCGCCTGCGC TAAGTCGCGA GCCAGGTTTA AC -            #CGTTGCGT   1020                                                                 - - CACCGGGACC CGAGCCCCCG CGATGCCCTG GGGGCCGTGC TCACTACCAA AT -            #GTTAATAA   1080                                                                 - - AGCCCGCGTC TGTGCAAAAA AAAAA          - #                  - #                 1105                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 440 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: LATRTUT02                                                        (B) CLONE: 1352286                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - Met Arg Val Val Thr Ile Val Ile Leu Leu Cy - #s Phe Cys Lys Ala Ala       1               5  - #                10  - #                15               - - Glu Leu Arg Lys Ala Ser Pro Gly Ser Val Ar - #g Ser Arg Val Asn His                  20      - #            25      - #            30                   - - Gly Arg Ala Gly Gly Gly Arg Arg Gly Ser As - #n Pro Val Lys Arg Tyr              35          - #        40          - #        45                       - - Ala Pro Gly Leu Pro Cys Asp Val Tyr Thr Ty - #r Leu His Glu Lys Tyr          50              - #    55              - #    60                           - - Leu Asp Cys Gln Glu Arg Lys Leu Val Tyr Va - #l Leu Pro Gly Trp Pro      65                  - #70                  - #75                  - #80        - - Gln Asp Leu Leu His Met Leu Leu Ala Arg As - #n Lys Ile Arg Thr Leu                      85  - #                90  - #                95               - - Lys Asn Asn Met Phe Ser Lys Phe Lys Lys Le - #u Lys Ser Leu Asp Leu                  100      - #           105      - #           110                  - - Gln Gln Asn Glu Ile Ser Lys Ile Glu Ser Gl - #u Ala Phe Phe Gly Leu              115          - #       120          - #       125                      - - Asn Lys Leu Thr Thr Leu Leu Leu Gln His As - #n Gln Ile Lys Val Leu          130              - #   135              - #   140                          - - Thr Glu Glu Val Phe Ile Tyr Thr Pro Leu Le - #u Ser Tyr Leu Arg Leu      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Tyr Asp Asn Pro Trp His Cys Thr Cys Glu Il - #e Glu Thr Leu Ile        Ser                                                                                             165  - #               170  - #               175             - - Met Leu Gln Ile Pro Arg Asn Arg Asn Leu Gl - #y Asn Tyr Ala Lys Cys                  180      - #           185      - #           190                  - - Glu Ser Pro Gln Glu Gln Lys Asn Lys Lys Le - #u Arg Gln Ile Lys Ser              195          - #       200          - #       205                      - - Glu Gln Leu Cys Asn Glu Glu Lys Glu Gln Le - #u Asp Pro Lys Pro Gln          210              - #   215              - #   220                          - - Val Ser Gly Arg Pro Pro Val Ile Lys Pro Gl - #u Val Asp Ser Thr Phe      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Cys His Asn Tyr Val Phe Pro Ile Gln Thr Le - #u Asp Cys Lys Arg        Lys                                                                                             245  - #               250  - #               255             - - Glu Leu Lys Lys Val Pro Asn Asn Ile Pro Pr - #o Asp Ile Val Lys Leu                  260      - #           265      - #           270                  - - Asp Leu Ser Tyr Asn Lys Ile Asn Gln Leu Ar - #g Pro Lys Glu Phe Glu              275          - #       280          - #       285                      - - Asp Val His Glu Leu Lys Lys Leu Asn Leu Se - #r Ser Asn Gly Ile Glu          290              - #   295              - #   300                          - - Phe Ile Asp Pro Ala Ala Phe Leu Gly Leu Th - #r His Leu Glu Glu Leu      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Asp Leu Ser Asn Asn Ser Leu Gln Asn Phe As - #p Tyr Gly Val Leu        Glu                                                                                             325  - #               330  - #               335             - - Asp Leu Tyr Phe Leu Lys Leu Leu Trp Leu Ar - #g Asp Asn Pro Trp Arg                  340      - #           345      - #           350                  - - Cys Asp Tyr Asn Ile His Tyr Leu Tyr Tyr Tr - #p Leu Lys His His Tyr              355          - #       360          - #       365                      - - Asn Val His Phe Asn Gly Leu Glu Cys Lys Th - #r Pro Glu Glu Tyr Lys          370              - #   375              - #   380                          - - Gly Trp Ser Val Gly Lys Tyr Ile Arg Ser Ty - #r Tyr Glu Glu Cys Pro      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Lys Asp Lys Leu Pro Ala Tyr Pro Glu Ser Ph - #e Asp Gln Asp Thr        Glu                                                                                             405  - #               410  - #               415             - - Asp Asp Glu Trp Glu Lys Lys His Arg Asp Hi - #s Thr Ala Lys Lys Gln                  420      - #           425      - #           430                  - - Ser Val Ile Ile Thr Ile Val Gly                                                  435          - #       440                                             - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2082 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: LATRTUT02                                                        (B) CLONE: 1352286                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - GAATGCAGCC CATTCTCTGG AGAACTTCCT CACACACCGC AGCAAAGAGA AG -             #ACTGAAAG     60                                                                 - - ACAAACCTGG GTGCAGCCAG AGAGGTCCAG ATAGATGAGC TTGTGGCATC CA -            #TTCCCCAA    120                                                                 - - GTTCAGCCTA GGGACTCCAC GTACCCCAGC TGGGTCTCAT TGTTCCAGAA CT -            #GCATTAGT    180                                                                 - - TAAGATTACC CAGACTTGGA TTTCAAAGGA ATACTTTCAT TGTTCCGTCT GT -            #AACACGAA    240                                                                 - - GTAATTGGGG CCAGCTGGAT GTCAGGATGC GTGTGGTTAC CATTGTAATC TT -            #GCTCTGCT    300                                                                 - - TTTGCAAAGC GGCTGAGCTG CGCAAAGCAA GCCCAGGCAG TGTGAGAAGC CG -            #AGTGAATC    360                                                                 - - ATGGCCGGGC GGGTGGAGGC CGGAGAGGCT CCAACCCGGT CAAACGCTAC GC -            #ACCAGGCC    420                                                                 - - TCCCGTGTGA CGTGTACACA TATCTCCATG AGAAATACTT AGATTGTCAA GA -            #AAGAAAAT    480                                                                 - - TAGTTTATGT GCTGCCTGGT TGGCCTCAGG ATTTGCTGCA CATGCTGCTA GC -            #AAGAAACA    540                                                                 - - AGATCCGCAC ATTGAAGAAC AACATGTTTT CCAAGTTTAA AAAGCTGAAA AG -            #CCTGGATC    600                                                                 - - TGCAGCAGAA TGAGATCTCT AAAATTGAGA GTGAGGCGTT CTTTGGTTTA AA -            #CAAACTCA    660                                                                 - - CCACCCTCTT ACTGCAGCAC AACCAGATCA AAGTCTTGAC GGAGGAAGTG TT -            #CATTTACA    720                                                                 - - CACCTCTCTT GAGCTACCTG CGTCTTTATG ACAACCCCTG GCACTGTACT TG -            #TGAGATAG    780                                                                 - - AAACGCTTAT TTCAATGTTG CAGATTCCCA GGAACCGGAA TTTGGGGAAC TA -            #CGCCAAGT    840                                                                 - - GTGAAAGTCC ACAAGAACAA AAAAATAAAA AACTGCGGCA GATAAAATCT GA -            #ACAGTTGT    900                                                                 - - GTAATGAAGA AAAGGAACAA TTGGACCCGA AACCCCAAGT GTCAGGGAGA CC -            #CCCAGTCA    960                                                                 - - TCAAGCCTGA GGTGGACTCA ACTTTTTGCC ACAATTATGT GTTTCCCATA CA -            #AACACTGG   1020                                                                 - - ACTGCAAAAG GAAAGAGTTG AAAAAAGTGC CAAACAACAT CCCTCCAGAT AT -            #TGTTAAAC   1080                                                                 - - TTGACTTGTC ATACAATAAA ATCAACCAAC TTCGACCCAA GGAATTTGAA GA -            #TGTTCATG   1140                                                                 - - AGCTGAAGAA ATTAAACCTC AGCAGCAATG GCATTGAATT CATCGATCCT GC -            #CGCTTTTT   1200                                                                 - - TAGGGCTCAC ACATTTAGAA GAATTAGATT TATCAAACAA CAGTCTGCAA AA -            #CTTTGACT   1260                                                                 - - ATGGCGTATT AGAAGACTTG TATTTTTTGA AACTCTTGTG GCTCAGAGAT AA -            #CCCTTGGA   1320                                                                 - - GATGTGACTA CAACATTCAC TACCTCTACT ACTGGTTAAA GCACCACTAC AA -            #TGTCCATT   1380                                                                 - - TTAATGGCCT GGAATGCAAA ACGCCTGAAG AATACAAAGG ATGGTCTGTG GG -            #AAAATATA   1440                                                                 - - TTAGAAGTTA CTATGAAGAA TGCCCCAAAG ACAAGTTACC AGCATATCCT GA -            #GTCATTTG   1500                                                                 - - ACCAAGACAC AGAAGATGAT GAATGGGAAA AAAAACATAG AGATCACACC GC -            #AAAGAAGC   1560                                                                 - - AAAGCGTAAT AATTACTATA GTAGGATAAG GTAGAAATTG TTCTGATTGT AA -            #TTAGTTTT   1620                                                                 - - GTATTTTCTA TACTGGTGTT AGAAAACATA TGTTTACATT TGATTAACTG TG -            #TTGCCTAT   1680                                                                 - - TTATGCAGGG TAATCCAGCT AAAGGAAGCT TTCTTTAATT ATAAGTATTA TT -            #GTGACTAT   1740                                                                 - - TATAGTAATC AAGAGAATGC TATCATCCTG CTTGCCTGTC CATTTGTGGA AC -            #AGCATCTG   1800                                                                 - - GTGATATGCA ATTCCACACT GGTAACCTGC AGCAGTTGGG TCCTAATGAT GG -            #CATTAGAC   1860                                                                 - - TTTCATAATG TCCTGTATAA ATGTTTTTAC TGCTTTTAGA AAATAAAGAA AA -            #AAAACTTG   1920                                                                 - - GTTCATGTTT ACATGCCTTT CGATAGCTGT TTGTGCATAC TTAAAGATGA TC -            #AAAATGAT   1980                                                                 - - TTTATACAAA TGCTGTTATA ATAAAATGTC ATTCCCTACC CCTCTACTTT TT -            #TTCAGTAA   2040                                                                 - - GTCATCTTAT ACATTAAATA AATTTCCATT TCTGAAAAAA AA    - #                      - #2082                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 469 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: OVARTUT01                                                        (B) CLONE: 815087                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - Met Asp Val Glu Asn Glu Gln Ile Leu Asn Va - #l Asn Pro Ala Asp Pro       1               5  - #                10  - #                15               - - Asp Asn Leu Ser Asp Ser Leu Phe Ser Gly As - #p Glu Glu Asn Ala Gly                  20      - #            25      - #            30                   - - Thr Glu Glu Val Lys Asn Glu Ile Asn Gly As - #n Trp Ile Ser Ala Ser              35          - #        40          - #        45                       - - Ser Ile Asn Glu Ala Arg Ile Asn Ala Lys Al - #a Lys Arg Arg Leu Arg          50              - #    55              - #    60                           - - Lys Asn Ser Ser Arg Asp Ser Gly Arg Gly As - #p Ser Val Ser Asp Ser      65                  - #70                  - #75                  - #80        - - Gly Ser Asp Ala Leu Arg Ser Gly Leu Thr Va - #l Pro Thr Ser Pro Lys                      85  - #                90  - #                95               - - Gly Arg Leu Leu Asp Arg Arg Ser Arg Ser Gl - #y Lys Gly Arg Gly Leu                  100      - #           105      - #           110                  - - Pro Lys Lys Gly Gly Ala Gly Gly Lys Gly Va - #l Trp Gly Thr Pro Gly              115          - #       120          - #       125                      - - Gln Val Tyr Asp Val Glu Glu Val Asp Val Ly - #s Asp Pro Asn Tyr Asp          130              - #   135              - #   140                          - - Asp Asp Gln Glu Asn Cys Val Tyr Glu Thr Va - #l Val Leu Pro Leu Asp      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Glu Arg Ala Phe Glu Lys Thr Leu Thr Pro Il - #e Ile Gln Glu Tyr        Phe                                                                                             165  - #               170  - #               175             - - Glu His Gly Asp Thr Asn Glu Val Ala Glu Me - #t Leu Arg Asp Leu Asn                  180      - #           185      - #           190                  - - Leu Gly Glu Met Lys Ser Gly Val Pro Val Le - #u Ala Val Ser Leu Ala              195          - #       200          - #       205                      - - Leu Glu Gly Lys Ala Ser His Arg Glu Met Th - #r Ser Lys Leu Leu Ser          210              - #   215              - #   220                          - - Asp Leu Cys Gly Thr Val Met Ser Thr Thr As - #p Val Glu Lys Ser Phe      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Asp Lys Leu Leu Lys Asp Leu Pro Glu Leu Al - #a Leu Asp Thr Pro        Arg                                                                                             245  - #               250  - #               255             - - Ala Pro Gln Leu Val Gly Gln Phe Ile Ala Ar - #g Ala Val Gly Asp Gly                  260      - #           265      - #           270                  - - Ile Leu Cys Asn Thr Tyr Ile Asp Ser Tyr Ly - #s Gly Thr Val Asp Cys              275          - #       280          - #       285                      - - Val Gln Ala Arg Ala Ala Leu Asp Lys Ala Th - #r Val Leu Leu Ser Met          290              - #   295              - #   300                          - - Ser Lys Gly Gly Lys Arg Lys Asp Ser Val Tr - #p Gly Ser Gly Gly Gly      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Gln Gln Ser Val Asn His Leu Val Lys Glu Il - #e Asp Met Leu Leu        Lys                                                                                             325  - #               330  - #               335             - - Glu Tyr Leu Leu Ser Gly Asp Ile Ser Glu Al - #a Glu His Cys Leu Lys                  340      - #           345      - #           350                  - - Glu Leu Glu Val Pro His Phe His His Glu Le - #u Val Tyr Glu Ala Ile              355          - #       360          - #       365                      - - Ile Met Val Leu Glu Ser Thr Gly Glu Ser Th - #r Phe Lys Met Ile Leu          370              - #   375              - #   380                          - - Asp Leu Leu Lys Ser Leu Trp Lys Ser Ser Th - #r Ile Thr Val Asp Gln      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Met Lys Arg Gly Tyr Glu Arg Ile Tyr Asn Gl - #u Ile Pro Asp Ile        Asn                                                                                             405  - #               410  - #               415             - - Leu Asp Val Pro His Ser Tyr Ser Val Leu Gl - #u Arg Phe Val Glu Glu                  420      - #           425      - #           430                  - - Cys Phe Gln Ala Gly Ile Ile Ser Lys Gln Le - #u Arg Asp Leu Cys Pro              435          - #       440          - #       445                      - - Ser Arg Gly Arg Lys Arg Phe Val Ser Glu Gl - #y Asp Gly Gly Arg Leu          450              - #   455              - #   460                          - - Lys Pro Glu Ser Tyr                                                      465                                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2395 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: OVARTUT01                                                        (B) CLONE: 815087                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - ACAGCTCGAG CTCGAGCCGC AAAACTGTCT GCAGACGTCA ATTTCGCCCC CC -             #TCCCCCTT     60                                                                 - - GTGAGAACTC GCTACGTAGC CAGCAACTGT GTAGTGTCTA CAAATGATGA AA -            #ACGATCAG    120                                                                 - - AAATGCGATT AGGTGTCGGG GAAAAAAGGG TTTCCCCTGT TTTTAACTTG TA -            #TTTTTACT    180                                                                 - - TTAATTGTTA CAATCTTGAT ATTCTTAACG TGACTTTTTT GGGAAACCAC CA -            #AGTGCTTT    240                                                                 - - TTAAGCAAGG AGTTACTGAT TCTGAAGGAA GATTTCCATT AGGTAATTTG TT -            #TAATCAGT    300                                                                 - - GCAAGCGAAA TTAAGGGAAA ATGGATGTAG AAAATGAGCA GATACTGAAT GT -            #AAACCCTG    360                                                                 - - CAGATCCTGA TAACTTAAGT GACTCTCTCT TTTCCGGTGA TGAAGAAAAT GC -            #TGGGACTG    420                                                                 - - AGGAAGTAAA GAATGAAATA AATGGAAATT GGATTTCAGC ATCCTCCATT AA -            #CGAAGCTA    480                                                                 - - GAATTAATGC CAAGGCAAAA AGGCGACTAA GGAAAAACTC ATCCCGGGAC TC -            #TGGCAGAG    540                                                                 - - GCGATTCGGT CAGCGACAGT GGGAGTGACG CCCTTAGAAG TGGATTAACT GT -            #GCCAACCA    600                                                                 - - GTCCAAAGGG AAGGTTGCTG GATAGGCGAT CCAGATCTGG GAAAGGAAGG GG -            #ACTACCAA    660                                                                 - - AGAAAGGTGG TGCAGGAGGC AAAGGTGTCT GGGGTACACC TGGACAGGTG TA -            #TGATGTGG    720                                                                 - - AGGAGGTGGA TGTGAAAGAT CCTAACTATG ATGATGACCA GGAGAACTGT GT -            #TTATGAAA    780                                                                 - - CTGTAGTTTT GCCTTTGGAT GAAAGGGCAT TTGAGAAGAC TTTAACACCA AT -            #CATACAGG    840                                                                 - - AATATTTTGA GCATGGAGAT ACTAATGAAG TTGCGGAAAT GTTAAGAGAT TT -            #AAATCTTG    900                                                                 - - GTGAAATGAA AAGTGGAGTA CCAGTGTTGG CAGTATCCTT AGCATTGGAG GG -            #GAAGGCTA    960                                                                 - - GTCATAGAGA GATGACATCT AAGCTTCTTT CTGACCTTTG TGGGACAGTA AT -            #GAGCACAA   1020                                                                 - - CTGATGTGGA AAAATCATTT GATAAATTGT TGAAAGATCT ACCTGAATTA GC -            #ACTGGATA   1080                                                                 - - CTCCTAGAGC ACCACAGTTG GTGGGCCAGT TTATTGCTAG AGCTGTTGGA GA -            #TGGAATTT   1140                                                                 - - TATGTAATAC CTATATTGAT AGTTACAAAG GAACTGTAGA TTGTGTGCAG GC -            #TAGAGCTG   1200                                                                 - - CTCTGGATAA GGCTACCGTG CTTCTGAGTA TGTCTAAAGG TGGAAAGCGT AA -            #AGATAGTG   1260                                                                 - - TGTGGGGCTC TGGAGGTGGG CAGCAATCTG TCAATCACCT TGTTAAAGAG AT -            #TGATATGC   1320                                                                 - - TGCTGAAAGA ATATTTACTC TCTGGAGACA TATCTGAAGC TGAACATTGC CT -            #TAAGGAAC   1380                                                                 - - TGGAAGTACC TCATTTTCAC CATGAGCTTG TATATGAAGC TATTATAATG GT -            #TTTAGAGT   1440                                                                 - - CAACTGGAGA AAGTACATTT AAGATGATTT TGGATTTATT AAAGTCCCTT TG -            #GAAGTCTT   1500                                                                 - - CTACCATTAC TGTAGACCAA ATGAAAAGAG GTTATGAGAG AATTTACAAT GA -            #AATTCCGG   1560                                                                 - - ACATTAATCT GGATGTCCCA CATTCATACT CTGTGCTGGA GCGGTTTGTA GA -            #AGAATGTT   1620                                                                 - - TTCAGGCTGG AATAATTTCC AAACAACTCA GAGATCTTTG TCCTTCAAGG GG -            #CAGAAAGC   1680                                                                 - - GTTTTGTAAG CGAAGGAGAT GGAGGTCGTC TTAAACCAGA GAGCTACTGA AT -            #ATAAGAAC   1740                                                                 - - TCTTGCAGTC TTAGATGTTA TAAAAATATA TATCTGAATT GTAAGAGTTG TT -            #AGCACAAG   1800                                                                 - - TTTTTTTTTT TTTTTTTTTT TAAGCACTTG TTTTGGGTAC AAGGCATTTC TG -            #ACATTTTA   1860                                                                 - - TAAACCTACA TTTAAGGGGA ATTTTTAAAG GAAATGTTTT TTCTTTTTTT TT -            #TGTTTTTC   1920                                                                 - - GAGGGGGCAA GGAGGGACAG AAAAGTAACC TCTTCTTAAG TGGAATATTC TA -            #ATAAGCTA   1980                                                                 - - CCTTTTGTAA GTGCCATGTT TATTATCTAA TCATTCCAAG TTTTGCATTG AT -            #GTCTGACT   2040                                                                 - - GCCACTCCTT TCTTTCAAGG ACAGTGTTTT TTGTAGTAAA ATCACTGGTT TA -            #TACAAAGC   2100                                                                 - - TTTATTTAGG GGGTAAAGTT AAGCTGCTAA AACCCCATGT TGGCTGCTGC TG -            #TTGAGATA   2160                                                                 - - CTGTGCTTTG GGAGTAAAAA AAGAAAGTTA TTTCTTTGTC TTAAAGAATT TT -            #TAAAAAAT   2220                                                                 - - TAGTCATGAG ACTTATTCAT CTTTCCAGGG AACATACTGA TTGGTCTTAA AA -            #GACTAGAC   2280                                                                 - - AGTTAAGTAA AAGGTGGCTG GAACATCTAT TTTTCTACAA AACTGGAAAA AT -            #GAACCTGG   2340                                                                 - - TTCTAGAAGA ATGTACACCA AAATAAAACA TGTGAAGCAG TATTGAAAAA AA - #AAA            2395                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 168 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: GenBank                                                          (B) CLONE: 1683637                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - Met Phe Gln Ile Pro Glu Phe Glu Pro Ser Gl - #u Gln Glu Asp Ser Ser       1               5  - #                10  - #                15               - - Ser Ala Glu Arg Gly Leu Gly Pro Ser Pro Al - #a Gly Asp Gly Pro Ser                  20      - #            25      - #            30                   - - Gly Ser Gly Lys His His Arg Gln Ala Pro Gl - #y Leu Leu Trp Asp Ala              35          - #        40          - #        45                       - - Ser His Gln Gln Glu Gln Pro Thr Ser Ser Se - #r His His Gly Gly Arg          50              - #    55              - #    60                           - - Trp Gly Cys Gly Asp Pro Glu Ser Pro Gln Le - #u Leu Pro Arg Gly Asp      65                  - #70                  - #75                  - #80        - - Gly Gly Arg Arg Arg Asp Gly Gly Gly Ala Gl - #n Pro Phe Arg Gly Arg                      85  - #                90  - #                95               - - Ser Arg Ser Ala Pro Pro Asn Leu Trp Ala Al - #a Gln Arg Tyr Gly Arg                  100      - #           105      - #           110                  - - Glu Leu Arg Arg Met Ser Asp Glu Phe Val As - #p Ser Phe Lys Lys Gly              115          - #       120          - #       125                      - - Leu Pro Arg Pro Lys Ser Ala Gly Thr Ala Th - #r Gln Met Arg Gln Ser          130              - #   135              - #   140                          - - Ser Ser Trp Thr Arg Val Phe Gln Ser Trp Tr - #p Asp Arg Asn Leu Gly      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Arg Gly Ser Ser Ala Pro Ser Gln                                                          165                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 313 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: GenBank                                                          (B) CLONE: 1236329                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - Met Arg Val Val Thr Ile Val Ile Leu Leu Cy - #s Phe Cys Lys Ala        Ala                                                                              1               5  - #                10  - #                15              - - Glu Leu Arg Lys Ala Ser Pro Gly Ser Val Ar - #g Ser Arg Val Asn His                  20      - #            25      - #            30                   - - Gly Arg Ala Gly Gly Gly Arg Arg Gly Ser As - #n Pro Val Lys Arg Tyr              35          - #        40          - #        45                       - - Ala Pro Gly Leu Pro Cys Asp Val Tyr Thr Ty - #r Leu His Glu Lys Tyr          50              - #    55              - #    60                           - - Leu Asp Cys Gln Glu Arg Lys Leu Val Tyr Va - #l Leu Pro Gly Trp Pro      65                  - #70                  - #75                  - #80        - - Gln Asp Leu Leu His Met Leu Leu Ala Arg As - #n Lys Ile Arg Thr Leu                      85  - #                90  - #                95               - - Lys Asn Asn Met Phe Ser Lys Phe Lys Lys Le - #u Lys Ser Leu Asp Leu                  100      - #           105      - #           110                  - - Gln Gln Asn Glu Ile Ser Lys Ile Glu Ser Gl - #u Ala Phe Phe Gly Leu              115          - #       120          - #       125                      - - Asn Lys Leu Thr Thr Leu Leu Leu Gln His As - #n Gln Ile Lys Val Leu          130              - #   135              - #   140                          - - Thr Glu Glu Val Phe Ile Tyr Thr Pro Leu Le - #u Ser Tyr Leu Arg Leu      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Tyr Asp Asn Pro Trp His Cys Thr Cys Glu Il - #e Glu Thr Leu Ile        Ser                                                                                             165  - #               170  - #               175             - - Met Leu Gln Ile Pro Arg Asn Arg Asn Leu Al - #a Asn Tyr Ala Lys Cys                  180      - #           185      - #           190                  - - Glu Ser Pro Gln Glu Gln Lys Asn Lys Lys Le - #u Arg Gln Ile Lys Ser              195          - #       200          - #       205                      - - Glu Gln Leu Cys Asn Glu Glu Glu Lys Glu Gl - #n Leu Asp Pro Lys Pro          210              - #   215              - #   220                          - - Gln Val Ser Gly Arg Pro Pro Val Ile Lys Pr - #o Glu Val Asp Ser Thr      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Phe Cys His Asn Tyr Val Phe Pro Ile Gln Th - #r Leu Asp Cys Lys        Arg                                                                                             245  - #               250  - #               255             - - Lys Glu Leu Lys Lys Val Pro Asn Asn Ile Pr - #o Pro Asp Ile Val Lys                  260      - #           265      - #           270                  - - Leu Asp Leu Ser Tyr Asn Lys Ile Asn Gln Le - #u Arg Pro Lys Glu Phe              275          - #       280          - #       285                      - - Glu Asp Val His Glu Leu Lys Lys Leu Asn Le - #u Ser Ser Asn Gly Ile          290              - #   295              - #   300                          - - Glu Phe Ile Asp Pro Gly Ser Leu Arg                                      305                 3 - #10                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 469 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: GenBank                                                          (B) CLONE: 1384078                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - Met Asp Ile Glu Asn Glu Gln Thr Leu Asn Va - #l Asn Pro Thr Asp Pro       1               5  - #                10  - #                15               - - Asp Asn Leu Ser Asp Ser Leu Phe Ser Gly As - #p Glu Glu Asn Ala Gly                  20      - #            25      - #            30                   - - Thr Glu Glu Ile Lys Asn Glu Ile Asn Gly As - #n Trp Ile Ser Ala Ser              35          - #        40          - #        45                       - - Thr Ile Asn Glu Ala Arg Ile Asn Ala Lys Al - #a Lys Arg Arg Leu Arg          50              - #    55              - #    60                           - - Lys Asn Ser Ser Arg Asp Ser Gly Arg Gly As - #p Ser Val Ser Asp Asn      65                  - #70                  - #75                  - #80        - - Gly Ser Glu Ala Val Arg Ser Gly Val Ala Va - #l Pro Thr Ser Pro Lys                      85  - #                90  - #                95               - - Gly Arg Leu Leu Asp Arg Arg Ser Arg Ser Gl - #y Lys Gly Arg Gly Leu                  100      - #           105      - #           110                  - - Pro Lys Lys Gly Gly Ala Gly Gly Lys Gly Va - #l Trp Gly Thr Pro Gly              115          - #       120          - #       125                      - - Gln Val Tyr Asp Val Glu Glu Val Asp Val Ly - #s Asp Pro Asn Tyr Asp          130              - #   135              - #   140                          - - Asp Asp Gln Glu Asn Cys Val Tyr Glu Thr Va - #l Val Leu Pro Leu Asp      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Glu Thr Ala Phe Glu Lys Thr Leu Thr Pro Il - #e Ile Gln Glu Tyr        Phe                                                                                             165  - #               170  - #               175             - - Glu His Gly Asp Thr Asn Glu Val Ala Glu Me - #t Leu Arg Asp Leu Asn                  180      - #           185      - #           190                  - - Leu Gly Glu Met Lys Ser Gly Val Pro Val Le - #u Ala Val Ser Leu Ala              195          - #       200          - #       205                      - - Leu Glu Gly Lys Ala Ser His Arg Glu Met Th - #r Ser Lys Leu Leu Ser          210              - #   215              - #   220                          - - Asp Leu Cys Gly Thr Val Met Ser Thr Asn As - #p Val Glu Lys Ser Phe      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Asp Lys Leu Leu Lys Asp Leu Pro Glu Leu Al - #a Leu Asp Thr Pro        Arg                                                                                             245  - #               250  - #               255             - - Ala Pro Gln Leu Val Gly Gln Phe Ile Ala Ar - #g Ala Val Gly Asp Gly                  260      - #           265      - #           270                  - - Ile Leu Cys Asn Thr Tyr Ile Asp Ser Tyr Ly - #s Gly Thr Val Asp Cys              275          - #       280          - #       285                      - - Val Gln Ala Arg Ala Ala Leu Asp Lys Ala Th - #r Val Leu Leu Ser Met          290              - #   295              - #   300                          - - Ser Lys Gly Gly Lys Arg Lys Asp Ser Val Tr - #p Gly Ser Gly Gly Gly      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Gln Gln Pro Val Asn His Leu Val Lys Glu Il - #e Asp Met Leu Leu        Lys                                                                                             325  - #               330  - #               335             - - Glu Tyr Leu Leu Ser Gly Asp Ile Ser Glu Al - #a Glu His Cys Leu Lys                  340      - #           345      - #           350                  - - Glu Leu Glu Val Pro His Phe His His Glu Le - #u Val Tyr Glu Ala Ile              355          - #       360          - #       365                      - - Val Met Val Leu Glu Ser Thr Gly Glu Ser Al - #a Phe Lys Met Ile Leu          370              - #   375              - #   380                          - - Asp Leu Leu Lys Ser Leu Trp Lys Ser Ser Th - #r Ile Thr Ile Asp Gln      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Met Lys Arg Gly Tyr Glu Arg Ile Tyr Asn Gl - #u Ile Pro Asp Ile        Asn                                                                                             405  - #               410  - #               415             - - Leu Asp Val Pro His Ser Tyr Ser Val Leu Gl - #u Arg Phe Val Glu Glu                  420      - #           425      - #           430                  - - Cys Phe Gln Ala Gly Ile Ile Ser Lys Gln Le - #u Arg Asp Leu Cys Pro              435          - #       440          - #       445                      - - Ser Arg Gly Arg Lys Arg Phe Val Ser Glu Gl - #y Asp Gly Gly Arg Leu          450              - #   455              - #   460                          - - Lys Pro Glu Ser Tyr                                                      465                                                                          __________________________________________________________________________

What is claimed is:
 1. An isolated and purified polynucleotide sequence encoding an APOP comprising SEQ ID NO:3 or SEQ ID NO:5.
 2. A composition comprising the polynucleotide sequence of claim
 1. 3. An isolated and purified polynucleotide sequence which is completely complementary to the polynucleotide sequence of claim
 1. 4. An isolated and purified polynucleotide sequence comprising SEQ ID NO:4 or SEQ ID NO:6.
 5. An isolated and purified polynucleotide sequence which is completely complementary to the polynucleotide sequence of claim
 4. 6. An expression vector containing the polynucleotide sequence of claim
 1. 7. A host cell containing the expression vector of claim
 6. 8. A method for producing a polypeptide comprising the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:5, the method comprising the steps of:a) culturing the host cell of claim 7 under conditions suitable for the expression of the polypeptide; and b) recovering the polypeptide from the host cell culture.
 9. A method for detecting a polynucleotide encoding APOP in a biological sample containing nucleic acids, the method comprising the steps of:(a) hybridizing the polynucleotide of claim 3 to at least one of the nucleic acids in the biological sample, thereby forming a hybridization complex; and (b) detecting the hybridization complex, wherein the presence of the hybridization complex correlates with the presence of a polynucleotide encoding APOP in the biological sample.
 10. The method of claim 9 wherein the nucleic acids of the biological sample are amplified by the polymerase chain reaction prior to hybridization. 